Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases

ABSTRACT

The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Qβ replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays. Such assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules. The invention permits replacement of an RNA, that is autocatalytically replicatable with an RNA replicase and employed as a reporter or label in prior art assays, such as nucleic acid probe hybridization assays or immunoassays, with a nucleic acid comprising a DNA segment with the same base sequence as the RNA. The invention also includes the methods of the invention with Mn +2 , Co +2 , or Zn +2  in the solutions in which the DDRP activity occurs.

This application is a continuation of U.S. application Ser. No.08/480,041 filed on Jun. 6, 1995, which issued as U.S. Pat. No.6,090,589, and which is a continuation-in-part of U.S. application Ser.No. 07/638,508 filed on Dec. 31,1990, entitled NUCLEIC ACIDAMPLIFICATION WITH DNA-DEPENDENT RNA POLYMERASE ACTIVITY OF RNAREPLICASES, now abondoned.

FIELD OF THE INVENTION

The present invention is directed to a method and a kit for amplifyingnucleic acid segments and detecting nucleic acid analyte in a testsample. More specifically, the present invention relates to amplifyingnucleic acid segments using the DNA-dependent RNA polymerase activity ofRNA-dependent RNA replicases, such as Qβ replicase, and detecting theproducts of such amplification.

BACKGROUND OF THE INVENTION

The ability to detect specific target nucleic acid analytes usingnucleic acid probe hybridization methods has many applications. Amongthese applications are diagnoses of infectious or genetic diseases orcancer in humans or other animals; identification of viral or microbialcontamination of cosmetics, food or water; and identification orcharacterization of, or discrimination among, individuals at the geneticlevel, for forensic or paternity testing in humans and breeding analysisand stock improvement in plants and animals. The basis for applicationsof nucleic acid probe hybridization methods is the ability of anoligonucleotide or nucleic-acid-fragment probe to hybridize, i.e., forma stable, double-stranded hybrid through complementary base-pairing,specifically with nucleic acid segments which have a particular sequenceand occur only in particular species, strains, individual organisms orcells taken from an organism.

One of the basic limitations in nucleic acid probe hybridization assayshas been the sensitivity of the assays, which depends on the ability ofa probe to bind to a target molecule and on the magnitude of signal thatis generated from each probe that binds to a target molecule and thatcan be detected in a time period available for detection. Knowndetection methods in the assays include methods dependent on signalgenerated from a probe, as from fluorescent moieties or radioactiveisotopes included in the probe, or an enzyme, such as an alkalinephosphatase or a peroxidase, linked to the probe and, after probehybridization and separation of hybridized from unhybridized probe,incubated with a specific substrate to produce a characteristic coloredproduct. However, the practical detection limit of these assays is about200,000 target molecules (3 femtomolar concentration in 100 μl), whichis not sufficiently sensitive for many applications. Much effort istherefore being expended in increasing the sensitivity of detectionsystems for nucleic acid probe hybridization assays.

A second area of research which is receiving significant attention isenhancement of sensitivity by, in effect, increasing the number oftarget molecules to be detected, i.e., by the amplification of a segmentof target nucleic acid to quantities sufficient to be readily detectableusing currently available signal-producing and signal-detection methods.The traditional method of obtaining increased quantities of targetmolecules in a sample has been to grow an organism with the targetmolecule under conditions which enrich for the organism using variousculturing methods. (Lennette, E. H., et al. (1985), Manual of ClinicMicrobiology, editors, American Society for Microbiology, Washington,D.C.; Gerhardt, P., et al. (1981), Manual of Methods for GeneralBacteriology, Editors, American Society for Microbiology, Washington,D.C.). Recent advances in increasing the number of target molecules in asample have focused on target-dependent increases in the number ofreporter molecules which can be derived from individual targetmolecules. Such a “reporter molecule” may or may not have the sequenceof a segment of the corresponding target molecule. One example of theserecent advances is amplification using the so-called “polymerase chainreaction” (“PCR”). With respect to PCR amplification, reference is madeto Current Protocols in Molecular Biology, Suppl. 4, Section 5, Unit3.17, which is incorporated herein by reference, for a basic descriptionof PCR. Other references which describe PCR include Erlich, H. A., (Ed.)1989, PCR Technology, Stockton Press; Erlich, H. A., et al. (1988),Nature 331:461-462; Mullis, K. B. and Faloona, F. A. (1987), Methods inEnzymology, 155:335-350; Saiki, R. K., et al. (1986), Nature324:163-166; Saiki, R. K., et al. (1988), Science 239:487-491; Saiki, R.K., et al. (1985), Science 230:1350-1354; U.S. Pat. No. 4,683,195 toMullis, et al.; and U.S. Pat. No. 4,683,202 to Mullis.

In PCR, the double-stranded target nucleic acid is thermally denaturedand hybridized with a pair of primers which flank the double-strandedsegment of interest in the target (one primer hybridizing to the 3′-endof each strand of this double-stranded segment) and then the primers areextended in a DNA polymerase-catalyzed extension reaction. Numerous(e.g., typically twenty-five) cycles of the denaturation, hybridizationand primer-extension process generate, for each target molecule in asample of nucleic acids, many copies of reporter molecules, which aredouble-stranded DNAs with the same nucleic acid sequence as a segment(usually of about 100-2000 base pairs) of the target molecule. In atwenty-five cycle PcR amplification, more than about 10⁶ reportersegments can be generated for each target molecule present initially ina sample. The PCR process is cumbersome because of the need to performmany cycles of the reaction, which usually require two or more hours forsufficient amplification. Additionally, the amplification process ismore time-consuming if it is carried out manually. Further, it can bequite expensive if automated equipment is used.

Another recently disclosed amplification process, called the“transcription-based amplification system” (“TAS”), uses primers whichcomprise segments for a promoter, which is recognized specifically by aDNA-dependent RNA polymerase which can produce quickly a large number oftranscripts from segments operably linked for transcription to thepromoter. Reference is made to Gingeras, T. R., et al., PCT PatentPublication No. WO 88/10315. Using suitable primers and primer-extensionreactions with a single-stranded target molecule (e.g., an RNA or onestrand of a double-stranded DNA) generates a double-stranded productwhich has a promoter operably linked for transcription to a pre-selectedsegment of the target molecule. Transcription of this product with aDNA-dependent RNA polymerase that recognizes the promoter produces, in asingle step, 10 to 1,000 copies of an RNA comprising a sequencecomplementary to that of the target segment (i.e., the preselectedsegment of target molecule). Two additional rounds of primer extensionusing a reverse transcriptase enzyme and the RNA copies made in theinitial transcription step produce CDNA copies which are ready foradditional amplification by transcription using the DNA-dependent RNApolymerase to yield RNA with the same sequence as the target segment oftarget molecule. Additional cycles of CDNA synthesis and transcriptioncan be performed. While TAS amplification, like PCR, makes a largenumber of reporter molecules (RNA in the case of TAS), which have thesame sequence as a segment of the target molecule or the sequencecomplementary thereto, and uses fewer steps than PCR to achieve the samelevel of amplification, TAS requires two more enzymatic reactions, i.e.,DNA-dependent RNA polymerase-catalyzed transcription and reversetranscription, and one or two more enzymes (DNA-dependent RNA polymeraseand, if not used for primer-dependent DNA extension, reversetranscriptase) than PCR. Additionally, no time savings in comparisonwith PCR is claimed.

A third amplification procedure, which entails a form of amplificationof label attached to a probe rather than amplification of a segment orsegments of target nucleic acid analyte, is based on the use of the Qβreplicase enzyme and its RNA-dependent RNA polymerase activity.Reference is made to Blumenthal, T. and G. G. Carmichael (1979), Ann.Rev. Biochem. 48:525-548; PCT Patent Publication No. WO 87/06270 andU.S. Pat. No. 4,957,858 to Chu, B., et al.; Feix, G. and H. Sano (1976),FEBS Letters 63:201-204; Kramer, F. R. and P. M. Lizardi (1989), Nature339:401-402; U.S. Pat. No. 4,786,600 to Kramer; Lizardi, P. M., et al.(1988), Biotechnology 6:1197-1202; and Schaffner, W., et al. (1977), J.Mol. Biol. 117:877-907 for a further description of this procedure. Inthe procedure, a replicative (sometimes referred to as “replicatable”)RNA molecule is covalently joined to a specific hybridizing probe (i.e.,a single-stranded nucleic acid with the sequence complementary of thatof a segment (“target segment”) of target nucleic acid analyte in asample). The probe may be a segment embedded within a recombinantreplicative RNA or attached to one of the ends of a replicative RNA. Theprobe-replicatable RNA complex hybridizes (by means of the probesegment) to target nucleic acid analyte in a sample, and the probe-RNAcomplexes that have hybridized are then separated from those that havenot, and the replicatable RNAs of the complexes that did hybridize totarget are then (typically after separation from probe segment if probesegment was not embedded in the replicatable RNA) amplifiedexponentially by incubation with Qβ replicase, which catalyzesautocatalytic replication of the replicatable RNA to produce up to 10⁹reporter molecules (replicatable RNAs) for each hybridized targetmolecule. Such amplification can be completed in 30 minutes (Lizardi, etal., supra).

The extreme specificity of Qβ replicase for RNAs with certain structuraland sequence requirements for catalysis of autocatalytic replicationassures that only the replicatable RNA associated with probes isamplified (Kramer and Lizardi, supra, 1989). Other advantages includethe speed of the reaction and the simplicity of manipulations. However,a disadvantage includes the need to use RNA as a reporter molecule. AnRNA of a given sequence is more expensive to manufacture and moresensitive to heat-stable nucleases than the DNA with the same sequence.In addition, except in cases where a probe segment can be embedded in areplicative RNA, the target segment is not amplified with the reportermolecules.

SUMMARY OF THE INVENTION

The present invention rests on the discovery of a DNA-dependent RNApolymerase (“DDRP”) activity of Qβ replicase, the enzyme which catalyzesreplication of the genome of the bacteriophage Qβ, and functionalequivalents thereof (e.g., other RNA-dependent RNA replicases that haveDDRP activity). The discovery of this DDRP acytivity allows the use ofsubstrates which comprise 2′-deoxyribonucleotides or analogs thereof,including DNA substrates, for amplification by Qβ replicase and theother replicases with DDRP activity.

The DDRP activity of an RNA replicase results in production of an RNA(or a polyribonucleotide in which, at some positions, ribonucleotideanalogs are present), that is autocatalytically replicatable by the RNAreplicase, from any substrate, which comprises a segment with thesequence of the autocatalytically replicatable RNA and which includes,within the segment with this autocatalytically replicatable sequence, a2′-deoxyribonucleotide or an analog thereof, such as a2′-deoxyriboalkylphosphonate, 2′-deoxyribophosphorothioate,2′-deoxyribophosphotriester, or 2′-deoxyribophosphoramidate. In asubstrate for the DDRP activity of an RNA replicase, the segment, whichacts as the template for synthesis, catalyzed by the replicase, of theauto-catalytically replicatable RNA, can be a segment which encompassesthe entire substrate (and, therefore, includes both the 3′-end and the5′-end of the substrate), a segment which includes the 3′-end but notthe 5′-end of the substrate, a segment which includes the 5′-end but notthe 3′-end of the substrate, or a segment embedded within the substrate(and, therefore, including neither the 3′-end nor the 5′-end of thesubstrate). The substrate can be linear or closed circular and may bepart of a double-stranded nucleic acid. The segment or the substrate mayconsist entirely of 2′-deoxyribonucleotides (i.e., a DNA segment orsubstrate, respectively). The substrates with which the DDRP activity isoperative are not limited to homopoly-2′-deoxyribonucleotides, such aspoly-dAs, with poly-dC segments at their 3′-ends, or RNAs with poly-dCsegments at their 3′-ends. See Feix and Sano (1976), supra.

In the methods of the present invention, the substrates for the DDRPactivity of RNA replicases are “complex” substrates. A “complex”substrate is one which is a closed circle, which does not have a free3′-end; or one which has a free 3′-end but wherein the segment, which isthe template for synthesis of an autocatalytically replicatable RNAcatalyzed by the DDRP activity, does not include the 3′-end; or onewhich has a free 3′-end and wherein the segment, which is the templatefor synthesis of an autocatalytically replicatable RNA catalyzed by theDDRP activity, includes the 3′-end but has a segment other than apoly-dC at the 3′-end; or one which has a free 3′-end and wherein thesegment, which is the template for synthesis of an autocatalyticallyreplicatable RNA catalyzed by the DDRP activity, includes the 3′-end andhas a poly-dC at its 3′-end but has, as the subsegment of said segment,other than the poly-dC at the 3′-end, a subsegment which comprises atleast one 2′-deoxyribonucleotide or analog thereof but is not anhomopoly-2′-deoxyribonucleotide. The segment, which is the template in acomplex substrate for synthesis of an autocatalytically replicatable RNAcatalyzed by the DDRP activity of an RNA replicase, is referred to as a“complex segment” or “complex template.” In the methods of theinvention, the “complex segments” comprise at least one2′-deoxyribonucleotide or analog thereof.

Reference herein to a “poly dC” means a segment of at least two dC's.

Reference herein to a “2′-deoxyribonucleotide” means one of the fourstandard 2′-deoxyribonucleotides.

Reference herein to a “2′-deoxyribonucleotide analog” means an analog ofa 2′-deoxyribonucleotide, which analog (i) has, as the base, the base ofthe 2′-deoxyribonucleotide or said base derivatized at a ring carbon oran amino nitrogen; and (ii) is other than the corresponding, standardribonucleotide (rA for dA, rC for dC, rG for dG, U for T). A2′-deoxyribonucleotide analog, that is part of a template for DDRPactivity by an RNA replicase in accordance with the invention, will berecognized by the replicase in the template to place the ribonucleotidewith the base, that is complementary to that of the2′-deoxyribonucleotide, in the corresponding position of theautocatalytically replicatable RNA made from the template via the DDRPactivity.

The RNA (or polyribonucleotide with one or more ribonucleotide analogs)made as a result of the DDRP activity of an RNA replicase isautocatalytically replicatable by the replicase (or anotherRNA-dependent RNA replicase which recognizes the RNA copies as templatesfor autocatalytic replication). Thus, a segment that is a template forthe DDRP activity of an RNA replicase, is amplified, in the presence ofthe replicase, the ribonucleoside 5′-triphosphates, and, possibly,analogs of certain of the ribonucleoside 5′-triphosphates, because RNA(or polyribonucleotide with one or more ribonucleotide analogs) that ismade in the synthesis catalyzed by the DDRP activity isautocatalytically replicated by the same replicase.

In its most general sense, then, the invention is a method foramplifying complex nucleic acid templates using the DNA-dependent RNApolymerase activity of RNA replicases, such as that of bacteriophage Qβ.The invention also entails numerous applications of this amplificationmethod in making, amplifying, detecting, sequencing or otherwisetreating a nucleic acid of interest. Thus, the amplification process canbe used to make large amounts of RNA, which, for example, can be used asa nucleic acid probe, converted to CDNA for cloning, detected as part ofa nucleic acid probe hybridization assay, or sequenced.

The amplification method of the invention can be employed with a sampleof nucleic acid in a target-dependent manner, such that anautocatalytically replicatable RNA which has, or comprises a segmentwith, a pre-determined sequence will be produced at a level detectableabove background in the amplification carried out with the sample onlyif a “target” segment of nucleic acid (i.e., a segment with apre-determined “target” sequence) is present in the sample. Thus, theinvention entails a method for target nucleic acid segment-directedamplification of a reporter nucleic acid molecule which comprises usingthe DDRP activity of Qβ replicase, or another replicase having DDRPactivity. More specifically, to a sample of nucleic acid, one or morenucleic acid probes are added and the sample with the probes isprocessed such that a complex substrate for the DDRP activity of an RNAreplicase, such as Qβ replicase, occurs if and only if a target nucleicacid comprising one or more target segments occurs in the sample. Thiscomplex substrate is, or comprises, a complex segment which, in turn,comprises a pre-determined sequence (which is or comprises a reportersequence) and which is the template for the DDRP activity. Each of theprobes will hybridize to a target segment or the complement of a targetsegment and the probes will comprise segments such that, upon suitableprocessing, a nucleic acid that is the complex substrate for DDRPactivity can be made using the probes if and only if the targetsegment(s) is (are) present in the sample. Once the sample has beentreated so that substrate for DDRP activity occurs, if target nucleicacid is present, an RNA replicase, which has such activity with thesubstrate, is added, along with other reagents necessary for reactionscatalyzed by the replicase, to an aliquot of the sample. If targetnucleic acid was present, so that substrate for the DDRP activity wasproduced, autocatalytically replicatable RNA, which will have orcomprise the reporter sequence or the sequence complementary thereto,will be amplified to detectable levels by the DDRP activity coupled withthe autocatalytic replication of the RNA made with the DDRP activity. Iftarget nucleic acid was not present, no substrate for the DDRP activitywill be produced which comprises the reporter sequence and the replicasewill not yield autocatalytically replicated RNA with such reportersequence (or the sequence complementary thereto). RNA with such reportersequence serves as a “reporter” directly or, if further processed,indirectly. Thus, production of the RNA constitutes amplification of areporter molecule and the process is target-directed (i.e.,target-dependent).

The present invention provides methods for detecting the presence orabsence of a target nucleic acid analyte in a sample containing nucleicacid. These methods of the invention comprise target-directedamplification in accordance with the invention, with the DDRP activityof Qβ replicase, or other replicase with DDRP activity, of reporternucleic acid, and assay for reporter nucleic acid.

Among advantages provided by certain of the methods according to theinvention for detecting nucleic acid analyte is a reduction in thefrequency of “false positives” that occur in assays that employ suchmethods of the invention in comparison with assays that employ othermethods. This advantage is associated with the fact that DNA isamplifiable (more precisely, capable of initiating amplification) usingthe DDRP activity of Qβ and other replicases in connection with assaysystems, and DNAs can be modified in specific ways using enzymes whichdo not modify RNAs in the same ways, if at all.

Several different embodiments of the target-dependent amplificationmethods of the invention are provided. These embodiments depend ondifferent structures of, and methods of treating, the various nucleicacid probes employed to provide in a sample of nucleic acid, in a mannerdependent on the presence of target nucleic acid in the sample, acomplex nucleic acid segment which is amplifiable using the DDRPactivity of an RNA replicase.

In one embodiment, to a sample of nucleic acid, which may includenucleic acid comprising a pre-selected target segment, a nucleic acidprobe is added which comprises both a replicase-amplifiable, complexsegment, which, as indicated above, comprises at least one2′-deoxyribonucleotide, and an anti-target (“probing”) segment, whichhas the sequence complementary to that of the target segment. Thenucleic acid probe that hybridizes to target segment, if any, in thesample is separated from probe that did not hybridize and hybridizedprobe is treated under amplification conditions in the presence of Qβreplicase, or another replicase exhibiting a DDRP activity with thereplicase-amplifiable segment of the probe, resulting in thetarget-dependent production and amplification of reporter nucleic acidmolecules. The process may also include the step of determining whetheramplification has occurred.

In another embodiment, similar to that just described, the separation ofprobe hybridized to target from that not so hybridized is accomplishedby subjecting the nucleic acid of the sample, after hybridization ofprobe to any target that may be present, to the action of a nucleasethat will digest the replicase-amplifiable segment of any unhybridizedprobe. In this embodiment, in which probe, if hybridized, is protectedfrom digestion, as in other embodiments of target-dependentamplification processes in accordance with the invention, if theamplification process is part of an assay for target analyte, amplifiedmaterial will be tested for using any of the many methods known to theskilled.

In another embodiment, the target nucleic acid segment is hybridizedwith two probes in such a fashion that, after hybridization with thetarget nucleic acid, the 3′-end of the anti-target segment of one of theprobes will be adjacent to the 5′-end of the anti-target segment of theother probe. Each probe comprises a portion of a nanovariant DNA, orother amplifiable DNA, covalently linked to anti-target segment. In oneprobe, the anti-target segment is at the 5′-end and, in the other, atthe 3′-end. Once hybridized, the probes may be ligated via theanti-target segments. Preferably, T4 DNA ligase or another suitableligase is used for the ligation. After the ligation, if it is carriedout, or the hybridization, if ligation is not carried out, the adjacentprobes are amplified via the DDRP activity of Qβ replicase or afunctional equivalent thereof. If the ligation/amplification process is,for example, part of a nucleic acid probe hybridization assay method,then, once amplification has been carried out, the amplified material isdetected by a suitable means known to those skilled in the art. Theamplified RNA, which comprises the sequence of the joined anti-targetsegments or the complement of that sequence, is a recombinantautocatalytically replicatable RNA wherein a segment, corresponding tothe joined anti-target segments, is inserted into another RNA which isautocatalytically replicatable. Only if target segment was present inthe sample will amplified RNA, which comprises the sequence of thejoined anti-target segments or the complement of that sequence, beproduced in the amplification process.

Two DNA probes are also employed in another embodiment of the invention.A first probe, for use in accordance with the embodiment, has a 3′-endwhich is an anti-target segment complementary (or nearly complementary)in sequence to a first target segment of target nucleic acid and issuitable for priming DNA synthesis using target nucleic acid astemplate. A second probe, for use in the embodiment, has a 3′-end whichis a segment (termed a “target-like” segment) with the same (or nearlythe same) sequence as a second target segment of target nucleic acid andalso is suitable for priming DNA synthesis using the complement oftarget nucleic acid as template. The 3′-terminal nucleotide of saidsecond target segment is located 5′- from the 5′-terminal nucleotide ofsaid first target segment. Thus, the second probe can prime DNAsynthesis on the primer extension product of the first probe annealed totarget nucleic acid. The 5′-ends of both of the probes are replicaseamplifiable or parts of nucleic acid that is replicase amplifiable,e.g., 5′-end of a nanovariant (+) DNA at the 5′-end of the first probeand 5′-end of a corresponding nanovariant (−) DNA at the 5′-end of thesecond probe. In the amplification process, the first probe is annealedto target and extended and the resulting extension products arepreferably strand-separated by thermal denaturation, or if target isRNA, may be strand-separated by treatment with an enzyme providing RNaseH activity. To the strand of the extension product which comprises thefirst probe at the 5′-end, the second probe is annealed and extended.Subsequent to, or simultaneously with, extension of second probe,amplification is catalyzed with the DDRP activity of Qβ replicase orequivalent. If, but only if, target nucleic acid or its complement ispresent in a sample of nucleic acid with which this dual primerextension/amplification process is carried out, amplified product willinclude nucleic acid which comprises (i) the complement of theanti-target segment of the first probe, (ii) the target-like segment ofthe second probe, and (iii) the same segment, if any, between thesegments of (i) and (ii) as occurs between the two target segments intarget nucleic acid. Thus, a nucleic acid probe hybridization assaymethod of the invention is provided by following the dual primerextension/amplification process by any conventional assay for amplifiedproduct which comprises these two, or three, segments.

In another embodiment of the invention, a probe can be employed which isreferred to for convenience as an “RNA probe” but which either consistsentirely of ribonucleotides (and is an RNA probe) or comprises in itssequence a sufficient number of ribonucleotides to permit degradationwith a ribonuclease or chemical treatment that degrades RNA but DNA, ifat all, at a much slower rate. The RNA probe comprises an anti-targetsegment, which is complementary or nearly complementary in sequence to atarget segment, which is at the 3′-end of target nucleic acid or asegment thereof, so that target segment can prime DNA synthesis on theRNA probe as template. At its 5′-end the RNA probe comprises a replicaseamplifiable segment. The target nucleic acid is treated so that the3′-end of the target segment is “free,” i.e., its 3′-terminal nucleotidehas a 3′-hydroxyl and is at the end of a nucleic acid and not covalentlyjoined, except through its 5′-carbon, to another nucleotide. The free3′-end of target segment is preferably provided by any conventionaltechnique by treating target nucleic acid prior to annealing RNA probeto target (or part thereof). The probe, and any target in the system,are combined under hybridizing conditions, the target segment isextended in a primer-extension reaction catalyzed by the reversetranscriptase activity of an enzyme which has such activity, and the RNAin the system is then degraded chemically or using enzymes with RNaseactivities, as understood in the art. This degradation of RNA issufficiently extensive, when coupled with dilution that might also becarried out, to diminish the concentration of RNA probe that retains areplicase-amplifiable segment and that thereby is operative, as atemplate for amplification by the replicase to be employed subsequentlyin the process, to a sufficiently low level that amplification of anysuch probe that might remain in an aliquot of sample on whichamplification is carried out will not be observable. Typically, afterthe reverse transcription of the RNA probe, the solution (or an aliquotthereof) will be treated so that the concentration ofamplifiable-segment-retainng RNA in the aliquot of carried out will beless than 1/1000, and preferably less than 1/10,000, the concentrationof complex template for DDRP activity that will be present if targetsegment was present in the sample of nucleic acid to which theamplification process is applied. More preferably, degradation of RNAwill be coupled with dilution so that, statistically, less than onemolecule of RNA with an amplifiable segment remains in the aliquot onwhich amplification is carried out. Any DNA-extension product remainingafter target segment extension and RNA degradation comprises arepicase-amplifiable, complex DNA segment. After degradation of RNA inthe system, and substantial elimination of RNA-degrading conditions oractivities, the DDRP activity of Qβ replicase or a functional equivalentis employed to amplify the replicase amplifiable segment added to anytarget DNA. Amplification will occur only if target segment, capable ofpriming DNA extension reaction on RNA probe as template, was present ina sample being tested. Thus, by applying after the amplificationreaction any conventional method to test for the presence of amplifiedproduct, a method of assaying for target nucleic acid is also provided.

An RNA probe, which may consist entirely of ribonucleotides or comprisesin its sequence a sufficient number of ribonucleotides to permitdegradation with a ribonuclease or chemical treatment that degrades RNAbut not DNA, can be employed in another embodiment of the invention,wherein three probes are employed. The first probe, which can be DNA orRNA or chimeric (i.e., any combination of ribonucleotides and2′-deoxyribonucleotides or analogs of either), comprises at its 5′-end afirst anti-target segment with the sequence complementary to or nearlycomplementary to that of a first target segment of target nucleic acid.The first probe must hybridize to its corresponding target segment withsufficient stability to block chain-extension of a second probe, aspresently described. The second probe, which also can be DNA or RNA orchimeric, compromises a second anti-target segment at its 3′-end withthe sequence complementary to, or nearly complementary to, that of asecond target segment of target nucleic acid and, when annealed totarget nucleic acid, is capable of priming DNA synthesis, using targetnucleic acid as template. The 3′-end of the first target segment islocated 5′ from the 5′-end of the second target segment and is separatedfrom the 5′-end of the second target segment by a gap of at leastseveral, and up to about 2000, bases. The third probe is referred to forconvenience as an RNA probe but, like the RNA probe described above,which comprises a replicase-amplifiable segment, must only comprise asufficient number of ribonucleotides to be susceptible, throughprocesses which degrade RNA, to having eliminated thereplicase-amplifiability of its replicase-amplifiable segment. The thirdprobe comprises a target-like segment at its 3′-end and a replicaseamplifiable segment. The target-like segment has the same or nearly thesame sequence as a third segment of target nucleic acid, which comprisesat its 5′-end at least several nucleotides of the gap between the firstand second target segments (and may overlap the second target segment)and which has as its 5′-terminal base the base that is adjacent to the3′-terminal base of the first target segment. The third probe, throughthe target-like segment, must be capable of priming DNA synthesis usingas template the chain-extension product of the second probe, made usingtarget nucleic acid as template. To amplify a reporter segment inaccordance with the invention, in a target nucleic acid-dependentmanner, the nucleic acid of a sample is rendered single-stranded andfirst and second probes are added to the sample, which is subjected toconditions whereby the probes will anneal to target if present andsecond probe, once annealed, will be extended in a primer-directed,template-dependent DNA extension reaction catalyzed by an enzyme such asKlenow Fragment of E.coli DNA polymerase I. The extension added tosecond probe in this extension reaction will have the sequencecomplementary to that of the gap between the first and second targetsegments in target nucleic acid. After the extension reaction, thesample is treated to strand-separate (e.g., thermally denature) theextension product, and then subjected to conditions whereby the thirdprobe anneals to its target segment, which will comprise at least partof the 3′-end of the segment added to the 3′-end of second probe in theextension reaction and may overlap at least a part of the segment of theextension product which was second probe, and at least the extendedsecond probe is further extended, employing reverse transcriptaseactivity and the third probe, including its replicase amplifiablesegment, as template. Subsequent to the second extension of secondprobe, the sample is treated as described above, for the embodiment ofthe invention which utilizes an RNA probe, to substantially eliminatereplicase-amplifiable segment of third probe by diminishing theconcentration of such segment to an insignificant level before replicaseis added to effect amplification. Thus, the solution is subjected toconditions to degrade RNA chemically or enzymatically, as understood inthe art, and might be treated further to dilute remainingreplicase-amplifiable segment of third probe. After degradation of theRNA and substantial elimination of RNA-degrading conditions, Qβreplicase or another RNA replicase, which recognizes thereplicase-amplifiable segment of the RNA probe as a template forautocatalytic replication, is added to the sample and the sample issubjected to conditions whereby the DDRP activity of the replicasecatalyzes amplification from the complex, replicase-amplifiable segmentof doubly extended second probe. As in other embodiments of theinvention, once the DDRP activity-catalyzed amplification has occurred,the amplified material may be detected by suitable means known to thoseskilled in the art.

As the skilled will understand, target segment(s) is (are) selected sothat, in a sample of nucleic acid to which a method of the invention isapplied, target segment, or the combination of target segments, requiredfor amplification in accordance with the method of the invention tooccur is present in an amount distinguishable from background only iftarget nucleic acid is present in the sample. Preferably targetsegment(s) is (are) selected so that the required target segment orcombination of target segments is absent from a sample unless targetnucleic acid is present.

The present invention is also directed to quantification of the amountof target nucleic acid analyte in a sample. Quantification isaccomplished by comparing the amount detected of a first amplifiednucleic acid, the amplification of which occurs only if target nucleicacid analyte is present in a sample, with the amount detected of asecond amplified nucleic acid, the amplification of which is carried outin parallel with that of the first amplified nucleic acid and occurs onaccount of the presence in the sample of a preseledted nucleic acidwhich serves as an internal standard and is present in the sample in aknown amount.

The present invention also encompasses a test kit for detection of aspecific target nucleic acid analyte in a sample of nucleic acid. Thekit comprises one or more nucleic acid probes required foramplification, in accordance with the invention, of a reporter molecule,Qβ replicase or an equivalent enzyme to provide DDRP activity, and otherenzymes (if any) required for processing of analyte or probe(s) prior toor simultaneously with amplification catalyzed by the DDRP activity. Thekit may also comprise means for detecting reporter nucleic acid producedin the amplification according to the invention, and various components,such as buffers and nucleoside triphosphates, to facilitate carrying outthe required hybridizations and enzymatically catalyzed reactions,including autocatalytic replication. A kit may also comprise componentsrequired for amplification associated with a pre-selected nucleic acidas an internal standard and detection of product from suchamplification, in order to provide for quantification in accordance withthe invention of target nucleic acid analyte to be assayed for with thekit. The various components of kits according to the invention may bepackaged in a kit in any of a variety of ways, among usually a pluralityof vials or other containers, as dictated by factors understood in theart, such as the need to preserve the stability and purity of thecomponents over the shelf-life of the kit, the order in which variouscomponents are used in accordance with the invention, convenience inusing the kits, convenience and cost in manufacturing the kits, and thelike.

Various methods known in the art can be employed to detect reportermolecules provided by an amplification process in accordance with theinvention. Thus, the reporter nucleic acid can be reacted with variousdyes and the dye detected visually or spectrophotometrically.Alternatively, a ribonucleoside 5′-triphosphate, that is labelled fordetection and remains active as a substrate for the Qβ replicase orother replicase catalyzing the amplification, can be employed in theamplification reaction and then signal from the label incorporated intothe amplified reporter can be detected directly or, after association ofthe label with a signal-generating molecule, indirectly. In stillanother alternative, reporter nucleic acid resulting from amplificationcan be hybridized with nucleic acid probe that is labelled for detectionand signal associated with such probe hybridized to reporter can bedetected directly or indirectly.

Among the advantages of the methods of the invention, and kits of theinvention for carrying out the methods, is speed. The amplificationprocess of the invention is typically able to produce more than 10⁹reporter molecules for each target molecule in a sample in about 60minutes. Further, systems for carrying out the present invention arerelatively simple in design and superior to systems which require use ofRNA to initiate amplification. The DNA used for this purpose in methodsof the present invention is resistant to degradation catalyzed by RNasesand provides more synthetic options than their RNA counterparts.Chemically synthesized DNA also provides a cost advantage over RNA. Thepresent invention is especially useful for amplification based on a rarespecies of nucleic acid present in a mixture of nucleic acids to provideeffective detection of the presence, and quantity, of the species.

Target nucleic acid analytes for amplification or detection by themethods of the present invention include, inter alia, nucleic acidscharacteristic of bacteria, viruses and other vectors of humaninfectious diseases; genomic nucleic acids comprising abnormalitieswhich underlie human genetic diseases; genomic nucleic acids comprisinghuman cancer genes; nucleic acids used in forensic analyses, paternitytesting, compatibility testing for bone marrow transplantations,characterization of plants and animals using restriction fragment lengthpolymorphism, and correlations of improvements through animal-orplant-breeding with genetic changes; and nucleic acids characteristic oforganisms which contaminate foods, cosmetics or water or whose presenceis diagnostic of environmental conditions in the environment in whichthe organisms occur.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing illustrating a method of target nucleicacid segment-directed amplification of a reporter molecule byhybridization of a DNA probe to target sequence (target segment)followed by separation of the hybridized probe from the unhybridized andthen amplification of the amplifiable segment of the probe whilehybridized to target. As illustrated in the Figure, the amplifiedproduct (RNA) may be detected.

FIG. 2 is a schematic drawing illustrating alternate forms which a probecan take for use in the method described in FIG. 1.

FIG. 3 is a schematic drawing illustrating another aspect of the presentinvention for a target nucleic acid segment-directed amplification of areporter molecule. In the method illustrated in FIG. 3, probe ishybridized to target and then an enzyme with single-stranded 3′-to-5′single-stranded exonuclease activity is added to degrade any probe whichis not protected from degradation by being hybridized to target.Finally, protected, undegraded probe is amplified. As illustrated in theFigure, the amplified product may be detected.

FIG. 4 is a schematic drawing illustrating another aspect of the presentinvention for a target nucleic acid segment-directed amplification of areporter molecule. In the method illustrated in FIG. 4, two probes arehybridized to adjacent segments of target and ligated and then theresulting ligated probe is amplified. As illustrated in the Figure, theamplified product may be detected.

FIG. 5 is a schematic drawing illustrating another aspect of the presentinvention for a target nucleic acid segment-directed amplification of areporter molecule. In the method illustrated in FIG. 5, a first probe ishybridized to target and primes chain extension with target as template,the product of the chain extension is strand-separated, a second probeis hybridized to the extended first probe and primes chain extensionwith extended first probe as template. The product of this second chainextension comprises a replicase amplifiable segment which, in turn,comprises a segment which has the sequence of a segment of the target.Amplification is carried out with the product of the second chainextension. As illustrated in the Figure, the amplified product may bedetected.

FIG. 6 is a schematic drawing illustrating another aspect of the presentinvention for a target nucleic acid segment-directed amplification of areporter molecule. In the method illustrated in FIG. 6, an RNA probe isused and the target segment is at the 3′-end of target nucleic acid.After hybridization of probe to target, both are extended in chainextension reactions, then RNA is digested and amplification is carriedout with replicase-amplifiable DNA added to target in the chainextension of target. As illustrated in the Figure, the amplified productmay be detected.

FIG. 7 is a schematic drawing illustrating another aspect of the presentinvention for a target nucleic acid segment-directed amplification of areporter molecule. In the method as illustrated in FIG. 7, three probes,designated A, B and C, are employed. Probes A and B lackreplicase-amplifiable segments. Probe C is an RNA and comprises areplicase amplifiable segment. Probes A and B hybridize to targetsegments separated by a gap, the target segment for A being located 5′from that for B. The anti-target segment of C, which is actually a“target-like segment,” has the same sequence as the segment of targetadjacent to and immediately 3′ from the 3′-end of the target segment forA. Probes A and B are hybridized to target and at least B is chainextended to the 5′-end of A. The product of the chain extension isstrand-separated and probe C is hybridized to chain-extended B andanother chain extension is carried out, RNA is digested, andamplification is carried out beginning with the replicase amplifiablesegment added in the second chain extension of B. As illustrated in theFigure, amplified product may be detected.

FIG. 8 illustrates a partial restriction site and functional map of theplasmid pNV1-3-4 and the sequence of a fragment from the plasmid whichcomprises a probe used in a method according to the invention, asdescribed in Example 6.

FIG. 9(a) and FIG. 9(b) are graphs representing the results of anamplification and detection procedure according to the invention with anucleic acid (phage M13mp19 DNA), which comprises target segment forprobe, and a nucleic acid (phage φX174 DNA) which lacks target segmentfor probe.

FIG. 10 illustrates a partial restriction site and functional map ofplasmid pMDV XhoI and the sequence of the HindIII-EcoRI fragment of theplasmid. The HindIII-EcoRI fragment comprises a segment, the strands ofwhich are midivariant DNAs and which comprises an inserted segment withan XhoI site.

DETAILED DESCRIPTION OF THE INVENTION

The present invention rests on the surprising and unexpected discoverythat an RNA replicase, such as Qβ replicase, has DDRP activity with acomplex template which comprises a 2′-deoxyribonucleotide or an analogthereof in place of a ribonucleotide but otherwise has the sequence ofan RNA which is autocatalytically replicatable by the replicase. Asindicated hereinabove, the invention encompasses numerous practicalapplications of this discovery, in nucleic acid segment amplification,target nucleic acid detection, and other fields.

Thus, in one of its aspects, the invention entails a method ofamplifying a complex nucleic acid segment, which comprises a2′-deoxyribonucleotide or an analog thereof, and has the sequence of anRNA which is autocatalytically replicatable by an RNA replicase, whichmethod comprises subjecting a sample which comprises said segment toconditions effective for autocatalytic replication by said replicase.

“Complex nucleic acid segment” is defined above.

Conditions effective for autocatalytic replication by an RNA replicase,such as Qβ replicase, are well known or easily ascertained by theskilled. Such conditions entail providing in the aqueous solution, inwhich the replicase is present, conditions of pH, ionic strength,temperature, and concentration of Mg⁺² at which the replicase is activein catalyzing autocatalytic replication and providing as well in saidsolution the four ribonucleoside 5′-triphosphates (hereinafter referredto simply as “ribonucleoside triphosphates”), which RNA replicasesemploy as substrates in catalyzing the process. Examples of suchconditions are provided in the examples hereinbelow. “Autocatalyticreplication” is, as understood in the art, a process catalyzed by an RNAreplicase in which an RNA template is employed as a substrate, alongwith the four ribonucleoside triphosphates, to make an RNA with thesequence complementary to that of the template. The RNA that is made isalso a template for the process. (Certain ribonucleoside triphosphateanalogs, such as rTTP or UTP with the 5-carbon of the uracil linked tobiotin (see, e.g., Langer et al., Proc. Natl. Acad. Sci. (1981) 78,6633) can be employed together with the four standard ribonucleosidetriphosphates in autocatalytic replication.) Usually, in a template forDDRP activity of a replicase in accordance with the invention, fewerthan 1 in 10 nucleotides will be a 2′-deoxyribonucleotide analog or aribonucleotide analog. Further, in carrying out DDRP activity on acomplex template and autocatalytic replication of the polynucleotideresulting from the DDRP activity, usually less than about 10 mole % ofsubstrate for the replicase for incorporation into the product of theautocatalytic replication will be analogs of ribonucleosidetriphosphates and, more typically, such analogs will be of only one ofthe four ribonucleoside triphosphates and will be present at less thanabout 10 mole % of that particular ribonucleoside triphosphate.Preferably, only 2′-deoxyribonucleotides and ribonucleotides will bepresent in templates for DDRP activity of a replicase and onlyribonucleoside triphosphates will be used as substrates for DDRPactivity and autocatalytic replication.

As indicated above, divalent transition metal ions, such as Mn⁺², Co⁺²,or Zn⁺², may also be present to advantage in reaction media in whichamplification via DDRP activity in accordance with the invention iscarried out. These ions, as well as the Mg⁺² required for replicaseactivity, are provided as any salt, which is sufficiently soluble in thesolution to achieve the desired metal ion concentration and the anion ofwhich does not inactivate the replicase. Suitable salts are well knownto the skilled and include the halide salts (e.g., chloride, bromide),the carbonates, the sulfates, the nitrates, and the like.

In another aspect, the invention entails applying the amplificationprocess of the invention in a target-dependent manner. Thus, theinvention entails a method of treating a sample comprising nucleic acidto make a reporter RNA, which is autocatalytically replicatable by anRNA replicase, only if the sample comprises a pre-selected targetnucleic acid segment, which method comprises (a) treating a firstaliquot of the sample of nucleic acid with one or more nucleic acidprobes, each of which is capable of hybridizing to a subsegment of thetarget segment or the complement of a subsegment of the target segment,provided that at least one of the probes is capable of hybridizing to asubsegment of the target segment, and which (i) if one probe is employedin the method, said probe is, or is capable of being processed to make,a complex nucleic acid segment comprising a 2′-deoxyribonucleotide or ananalog thereof and having the sequence of the reporter RNA or thecomplement of the reporter RNA, or (ii) if more than one probe isemployed in the method, said probes are capable of being processed tomake a complex or broken complex nucleic acid segment comprising a2′-deoxyribonucleotide or an analog thereof and having the sequence ofthe reporter RNA or the complement of the reporter RNA; (b) processingsaid first aliquot, including said probe or probes, to prepare a secondaliquot wherein (i) said complex or broken complex nucleic acid segmentcomprising a 2′-deoxyribonucleotide or an analog thereof and having thesequence of the reporter RNA or the complement thereof is made, if notprovided as part of a single probe, and remains in an amount that issignificant in view of step (c) only if target segment is present in thesample, and (ii) any nucleic acid segment, which lacks2′-deoxyribonucleotides and analogs thereof but is a template forsynthesis of reporter RNA or the complement thereof by said RNAreplicase, is reduced to an amount that is insignificant in view of step(c); and (c) subjecting the second aliquot, or a third aliquot takenfrom said second aliquot, to conditions effective for autocatalyticreplication in the presence of said replicase.

Several different embodiments of this target-dependent amplificationmethod of the invention are described elsewhere herein. The methodrequires production of reporter RNA (or its complement) via the DDRPactivity of a replicase. Because RNA (or RNA including ribonucleotideanalogs) made by DDRP activity is autocatalytically replicated, toprovide the RNA that is complementary in sequence, the “reporter RNA”can be selected, arbitrarily, to be either the RNA with the sequencecomplementary to that of the complex segment which is the template forthe DDRP activity or the complement of that RNA. To insure that thetarget-dependent amplification process, to the extent it is observable,is due to the DDRP activity of a replicase, which acts on a complexnucleic acid segment comprising a 2′-deoxyribonucleotide or analogthereof, any probe employed in the process, which provides a segmentwhich has the sequence of the reporter RNA or its complement, does notcomprise a 2′-deoxyribonucleotide or analog thereof, and is a templatefor the replicase (e.g., an RNA probe with such a segment) must bereduced to a level (e.g., concentration) that is insignificant, giventhe amplification process that is carried out on the complex segmentcomprising a 2′-deoxyribonucleotide or analog thereof, before thatamplification process is carried out. A level that is “insignificant”will vary depending on the details of the amplification process,including its duration and the rates of autocatalytic replication ofreporter RNA and its complement and DDRP activity using the complexsegment which is the template for such activity. A level is“insignificant” if it does not result in a measurable amount of reporterRNA when the process is carried out with a sample known to lack targetsegment. Preferably, of course, the level will be zero. Generally thislevel and, in any case, levels that are clearly “insignificant” areeasily achieved, as described elsewhere herein, by combining treatmentwith base (which, as well known, degrades RNA much more rapidly thanDNA) or a ribonuclease with dilution. (As a practical matter, the probesthat need to be reduced to an “insignificant” level are either RNAs orcomprise enough ribonucleotides to be susceptible to degradation byribonucleases.)

“Conditions effective for autocatalytic replication” require thatconditions not prevail which entail degradation at a substantial rate ofRNA, which is made by the DDRP activity and in the autocatalyticreplication. Thus, if ribonucleases might be present in step (c) of thetarget-dependent amplification process of the invention at a level whichmight cause problematic degradation of reporter RNA, ribonucleaseinhibitors should be employed to block such degradation. Such inhibitorsmight be employed in step (c) of the process if, in step (b), one ormore ribonucleases were employed to degrade probe that is a template forsynthesis of reporter RNA or complement thereof but is not a complexsegment comprising a 2′-deoxyribonucleotide or analog thereof.

Typically, one two or three probes, each of which is a DNA or an RNA,are employed in the target-dependent amplification process of theinvention for each target segment. If one probe, which is a DNA orotherwise is a complex substrate comprising a 2′-deoxyribonucleotide oranalog thereof, is used, such a substrate does not need to be made butthe sample with the probe is processed, by methods well known to theart, so that probe will remain at a significant level (e.g.,concentration) only if target segment is present. By “significant” levelis meant a level that, given the details of the process of amplificationvia DDRP activity and autocatalytic replication in part (c) of theprocess, yields an amount of reporter RNA that is observable (detectableabove “background”). If a single RNA probe, or more than one probe, areused, additional processing is required, as described in more detailelsewhere in the present specification, to make a complex segment, thatcomprises a 2′-deoxyribonucleotide or analog thereof and that is asubstrate for DDRP activity of the replicase, and have that segmentremain at a. significant level only if target segment is present and toreduce RNA probe (if any) to an insignificant level.

In still a further aspect, the invention entails a method of assaying asample for the presence of a target nucleic acid analyte which methodcomprises carrying out with the sample target nucleic acid-mediatedamplification, in accordance with the invention, of a reporter nucleicacid followed by assay of the sample for the presence of a nucleic acidwith the sequence of reporter nucleic acid (or its complement). The“reporter nucleic acid” is generally reporter RNA.

Thus, the invention entails a method of detecting the presence of atarget nucleic acid analyte, comprising a pre-selected target segment,in a test sample thought to contain said target nucleic acid, saidmethod comprising treating said sample of nucleic acid to make areporter RNA, which is autocatalytically replicatable by an RNAreplicase, only if the sample comprises said pre-selected targetsegment, and assaying for any reporter RNA so made, said treatingcomprising (a) treating a first aliquot of the sample of nucleic acidwith one or more nucleic acid probes, each of which is capable ofhybridizing to a subsegment of the target segment or the complement of asubsegment of the target segment, provided that at least one of theprobes is capable of hybridizing to a subsegment of the target segment,and which (i) if one probe is employed in the method, said probe is, oris capable of being processed to make, a complex nucleic acid segmentcomprising a 2′-deoxyribonucleotide or an analog thereof and having thesequence of the reporter RNA or the complement of the reporter RNA, or(ii) if more than one probe is employed in the method, said probes arecapable of being processed to make a complex or broken complex nucleicacid segment comprising a 2′-deoxyribonucleotide or an analog thereofand having the sequence of the reporter RNA or the complement of thereporter RNA; (b) processing said first aliquot, including said probe orprobes, to prepare a second aliquot wherein (i) said complex or brokencomplex nucleic acid segment comprising a 2′-deoxyribonucleotide or ananalog thereof and having the sequence of the reporter RNA or thecomplement thereof is made, if not provided as part of a single probe,and remains in an amount that is significant in view of step (c) only iftarget segment is present in the sample, and (ii) any nucleic acidsegment, which lacks 2′-deoxyribonucleotides and analogs thereof but isa template for synthesis of reporter RNA or the complement thereof bysaid RNA replicase, is reduced to an amount that is insignificant inview of step (c); and (c) subjecting the second aliquot, or a thirdaliquot taken from said second aliquot, to conditions effective forautocatalytic replication in the presence of said replicase.

Any of numerous methods can be employed to assay for reporter RNA (orits complement). In situations where the mass of reporter RNA and itscomplement, if made, will be substantial fraction of the mass of nucleicacid present after the amplification, a nucleic acid-staining dye cansimply be added to an aliquot of sample in which the amplification wascarried out and the aliquot can be visualized to see whether staininghas occurred. Staining will occur and be observed only if target nucleicacid was present to lead to production of reporter RNA. Situations inwhich this simple staining technique can be applied include those where,after amplification, the stained reporter RNA and its complement arevisible in an aliquot of sample and the mass of such stained reporterRNA and complement exceeds by a factor of at least about two the mass ofother nucleic acid present in the sample after the amplification.

In situations where the amount of reporter RNA and its complement formedin the amplification process is too low to allow simple staining to beused to detect whether target nucleic acid was present in a sample, thenucleic acid of an aliquot of a sample, after the amplification processis carried out with the sample, can be separated by size, e.g.,electrophoretically, and then stained. Production in the amplificationprocess of nucleic acid of the size of reporter RNA and its complement,as detected by observing stained nucleic acid of that size in thesize-separated nucleic acid, indicates that target nucleic acid waspresent in the sample of nucleic acid being analyzed.

Alternatively, reporter RNA and its complement, that are made during theamplification process if target analyte is present in a sample beingassayed, can be labelled in the course of the amplification, e.g., byemploying some ³²P-labelled ribonucleoside triphosphate or biotinylatedUTP in the substrate for the replicase, and then the labelled reporterRNA or its complement, if they are made, can be detected via the labelas understood in the art. Prior to the detection process, labelledreporter and its complement (if any) must be separated from labelledribonucleoside triphosphate that was not incorporated into RNA duringthe amplification process, e.g., chromatographically, by hybridizationof reporter RNA or complement thereof to latex beads or magneticparticles to which single-stranded nucleic acids with sequencescomplementary to that of a segment of reporter or its complement arecovalently attached.

Still other methods of assaying for production of reporter RNA or itscomplement are by nucleic acid probe hybridization assays for either. Inthese assays, a nucleic acid that is labeled for detection and that iscapable of hybridizing to the reporter RNA or its complement is employedas understood in the art.

Finally, because synthesis of reporter RNA and its complement in theamplification process consumes ribonucleoside triphosphates or analogsthereof, the concentration of such a compound in a solution in whichamplification will be occurring, if target analyte was present in asample being assayed, can be monitored to determine whetheramplification did occur. The depletion of such a compound indicates thatamplification has occurred. One such compound whose depletion can bemonitored readily is ATP; such monitoring can be carried out bymeasuring, as understood in the art, bioluminescence as catalyzed by aluciferase, e.g. from a beetle such as P. pyralis.

The invention also involves kits for carrying out the various methods ofthe invention, particularly the target-dependent amplification methodsand the methods for detecting nucleic acid analyte.

Thus, the invention entails a kit for amplification, dependent on thepresence in a sample of nucleic acid of a nucleic acid comprising apre-selected target segment, of a reporter RNA, which isautocatalytically replicatable by an RNA replicase, said kit comprising,packaged together, a replicase-holding container and one or moreprobe-holding containers; said replicase-holding container holding areplicase solution which comprises an RNA replicase for which thereporter RNA is a template for autocatalytic replication; and saidprobe-holding container, if one, or each of said probe-holdingcontainers, if more than one, holding a probe solution comprising one ormore of the nucleic acid probes required for said amplification(“required probes”), provided that all of said required probes are heldin the one or more probe-holding containers which the kit comprises;said required probe or, if more than one, each of said required probesbeing capable of hybridizing to a subsegment of the target segment orthe complement of a subsegment of the target segment, provided that: (i)at least one of the probes is capable of hybridizing to a subsegment ofthe target segment; (ii) if there is one required probe, said probe is,or is capable of being processed to make, a complex nucleic acid segmentcomprising a 2′-deoxyribonucleotide or an analog thereof and having thesequence of the reporter RNA or the complement of the reporter RNA;(iii) if there is more than one required probe, said probes are capableof being processed to make a complex or broken complex nucleic acidsegment comprising a 2′-deoxyribonucleotide or an analog thereof andhaving the sequence of the reporter RNA or the complement of thereporter RNA. These kits of the invention may comprises additionally,packaged with the replicase-holding container and the one or moreprobe-holding containers, one or more enzyme-holding containers, each ofwhich holds a solution of an enzyme used in any processing of probesnecessary to make a complex or broken complex nucleic acid segmentcomprising a 2′-deoxyribonucleotide or an analog thereof and having. thesequence of the reporter RNA or the complement of the reporter RNA.

These kits of the invention may be test kits for detecting the presenceof a target nucleic acid analyte, comprising a pre-selected targetsegment, in a test sample thought to contain said analyte. Such testkits comprise additionally reagents for rendering detectable reporterRNA or complement thereof produced in the amplification carried out withthe components of the kit on an aliquot of the test sample if saidsample comprises said analyte. In the test kits, such reagents will beheld in detection-reagent-holding containers that are packaged togetherwith the replicase-holding, probe-holding and any enzyme-holdingcontainers. Such reagents, which might be included in a test kit,include, for example, a solution of a dye to stain nucleic acid, asolution of a nucleic acid probe that is labeled for detection and thatis capable of hybridizing to reporter RNA or complement thereof, or asolution of a beetle luciferase.

“Autocatalytic replication” and “autocatalytically replicatable” areterms known in the art. See, e.g., Chu, et al., PCT Patent PublicationNo. WO 87/06270, and references cited therein; Kramer, et al., U.S. Pat.No. 4,786,600. Under conditions where the concentration ofautocatalytically replicatable template RNA does not exceed that of thereplicase catalyzing autocatalytic replication, the process is anexponential one.

For purposes of the present invention, the term “connector sequence” or“connector segment” is intended to mean a nucleic acid segment which isnot all or part of an amplifiable (i.e., autocatalytically replicatable)segment and not an anti-target segment, but rather is a segment thatjoins two of such segments in a probe.

The term “target nucleic acid,” as used herein, refers to the specificnucleic acid analyte to initiate a target-dependent amplification inaccordance with the invention or to be detected in a sample comprisingnucleic acid and suspected of containing the nucleic acid analyte. Atarget nucleic acid will comprise a “target segment,” to which probes ofthe invention hybridize in processes of the invention.

The term “anti-target nucleic acid sequence,” “anti-target sequence,”“anti-target” or “anti-target segment,” as used herein, is intended tomean the segment of a nucleic acid probe with a sequence (of bases)which is at least partially (and preferably exactly) complementary tothe sequence (of bases) of the nucleic acid segment (“target segment”)to which the probe is intended to hybridize in processes of theinvention. Hybridization between anti-target segments and theircorresponding target segments provides specificity for target nucleicacid in methods of the invention.

To effect hybridizations with the intended specificity in carrying outthe methods of the present invention generally requires that anti-targetsegments have at least six, and preferably at least 12, and morepreferably about 20-about 35 nucleotides. Factors which affectspecificity of hybridizations are well understood by the skilled andinclude, in addition to the lengths of the hybridizing segments, thecomplexity of the mixtures of nucleic acids in which the hybridizationare carried out and the stringency at which the hybridizations arecarried out. For a mixture of nucleic acid of a given complexity, theskilled can manipulate anti-target segment length, stringency, andselection of sequence of target segment to achieve the requiredspecificity.

The term “probe” or “nucleic acid probe” as used herein refers to anucleic acid which is a DNA, an RNA, or a chimeric nucleic acid andwhich comprises an anti-target segment. A probe may be madesynthetically, as in an automated synthesizer, or derived from cellularor viral substituents. It will be single-stranded, but may beaccompanied by its complementary strand (or a segment thereof). A probemust comprise an anti-target segment, However, as indicated above, insome embodiments of the invention a probe may be employed which isintended to hybridize to a segment which is complementary in sequence toa segment of target nucleic acid; the anti-target segment of such aprobe will be a “target-like” segment and, as such, will have the same,or nearly the same, sequence as a segment of target nucleic acid.

The term “reporter molecule” or “reporter nucleic acid” as used hereinis intended to mean a nucleic acid generated in an amplification processof the invention, which depends on the presence in a sample of a targetnucleic acid. “Reporter RNA” may consist of the four standardribonucleotides (unlabelled or labelled with a radioactive isotope(e.g., ³²P) of an element which occurs normally in the ribonucleotide)or, as described elsewhere herein, may include various ribonucleotideanalogs which function as substrates for an RNA replicase inautocatalytic replication and which, if present in an RNA with thesequence of a template for autocatalytic replication by an RNAreplicase, do not block the replicase from replicating the template.

“Amplification of a reporter nucleic acid” can mean either (i)replication by DDRP activity of a replicase of a complex reportersegment which comprises a 2′-deoxyribo-nucleotide or an analog thereofinto another reporter of complementary sequence which is completely anRNA (or which may comprise a ribonucleotide analog (e.g. uridine linkedthrough the 5-position of the uracil to a biotin moiety) the5′-triphosphate of which is a substrate for the replicase and which, inthe RNA, does not block the RNA's functioning as a template forautocatalytic replication by the replicase) and which is a template forautocatalytic replication by the replicase followed by autocatalyticreplication of said autocatalytically replicatable reporter or (ii)simply autocatalytic replication of an autocatalytically replicatablereporter which is an RNA or an RNA which comprises a ribonucleotideanalog as just described.

Either the presence or absence of reporter molecules, or the amountproduced, can be used as an indicator of the presence (or absence,respectively) of the target analyte in a sample. A “reporter molecule”will have a “reporter sequence,” through which the reporter molecule maybe detected and which may be the sequence of the entire molecule or asegment thereof.

“Amplification” of a nucleic acid or segment thereof means the processof making multiple copies of a nucleic acid which has the same sequenceas the nucleic acid (or segment) being amplified. The term“segment-directed amplification” or “target-directed amplification” asused herein is intended to mean a replicase-mediated process wherebyeach target nucleic acid molecule (or, more precisely, targetsegment(s), selected to be uniquely characteristic of target nucleicacid) is used to initiate production of multiple reporter molecules.

The term “amplifiable sequence” or “amplifiable nucleic acid sequence”is intended to mean the sequence of an RNA which is autocatalyticallyreplicatable by an RNA replicase. Thus, reference is sometimes made to a“replicase-amplifiable” sequence or segment.

A “segment” of a nucleic acid strand is the entire nucleic acid strandor any part thereof with a continuous sequence of at least twonucleotides (or nucleotide analogs) as in the nucleic acid strand. A“subsegment” of a segment of a nucleic acid strand is the entire segmentor any part of the segment with a continuous sequence of at least twonucleotides (or nucleotide analogs) as in the segment.

An “aliquot” of a sample or, more typically, a solution is, asunderstood by the skilled, a part of the sample or solution which willhave intrinsic properties (e.g., composition, concentrations ofconstituents) that are indistinguishable from those of the sample orsolution as a whole.

The discovery underlying the invention is that a nucleic acid whichcomprises a 2′-deoxyribonucleotide or analog thereof but has a complex,amplifiable sequence for an RNA replicase functions as a template forsynthesis, catalyzed by the replicase, of an RNA, which has thecomplementary, also amplifiable sequence, and which, therefore, isautocatalytically replicatable. Thus, surprisingly and advantageously, acomplex segment comprising a 2′-deoxyribonucleotide or an analog thereofand having an amplifiable sequence can be used to make anautocatalytically replicatable RNA and, thereby, initiate the process ofautocatalytic replication.

By reference herein to a nucleic acid which is an RNA is meant a nucleicacid consisting of only one or more of the four standardribonucleotides, adenosine monophosphate (A or rA), uridinemonophosphate (U), guanosine monophosphate (G or rG) and cytidinemonophosphate (C or rC). By reference herein to a “ribonucleotide,”without further qualification, is mean on of the four standardribonucleotides. By reference herein to a nucleic acid which is a DNA ismeant a nucleic acid consisting of only one or more of the four standard2′-deoxyribonucleotides, 2′-deoxyadenosine monophosphate (A or dA),2′-deoxythymidine monophosphate (T), 2′-deoxyguanosine monophosphate (Gor dG) and 2′-deoxycytidine monophosphate (C or dC). A “nucleotide”without further qualification means a 2′-deoxyribonucleotide, a2′-deoxyribonucleotide analog, a ribonucleotide, or ribonucleotideanalog. A “nucleic acid,” without further qualification, means adouble-stranded or single-stranded oligonucleotide or polynucleotide. A“chimeric” nucleic acid is a single-stranded nucleic acid in which someof the nucleotides are ribonucleotides or ribonucleotide analogs andsome are 2′-deoxyribonucleotides or 2′-deoxyribonucleotide analogs. A“non-analog” chimeric nucleic acid is a chimeric nucleic acid whichconsists of ribonucleotide(s).and 2′-deoxyribonucleotide(s) and, assuch, includes analogs of neither. An “hybrid” nucleic acid is adouble-stranded, or partially double-stranded, nucleic acid, in whichone of the strands is DNA and the other is RNA or chimeric or one of thestrands is RNA and the other is DNA or chimeric. The “nucleotide” at the5′-end of a nucleic acid need not necessarily have a single5′-phosphate; it might have, for example, a 5′-triphosphate or a5′-hydroxyl. Similarly, the “nucleotide” at the 3′-end of a nucleic acidneed not necessarily have a 3′-hydroxyl; it might for example, have a3′-phosphate.

2′-deoxyribonucleotide analogs, which may be included in complex nucleicacid templates for amplification via the DDRP activity of a RNAreplicase in accordance with the instant invention, are described above.

Among such 2′-deoxyribonucleotide analogs are “(2′-deoxyribonucleotide)phosphate analogs,” by which is meant analogs wherein the phosphate ofthe corresponding 2′-deoxyribonucleotide is replaced with a phosphateanalog such as an alkylphosphonate (wherein the alkyl group may be, forexample, a methyl, ethyl, n-propyl, or i-propyl), a phosphorothioate, aphosphotriester, or a phosphoramidate. Thus, analogs of2′-deoxyribonucleotides which may occur in complex templates for DDRPactivity of RNA replicases in accordance with the invention include2′-deoxyriboalkyl-phosphonates (see Blake et al., Biochemistry (1985)24, 6139), 2′-deoxyribophosphorothioates (see Froehler, TetrahedronLett. (1986) 27, 5575), 2′-deoxyribophosphotriesters (see Blackburtn etal., J. Chem. Soc. (C) (1966), 239), and 2′-deoxyribophosphoramidates(see Zwierzak, Synthesis (1975), 507).

A “ribonucleotide analog” is a ribonucleotide wherein the base isderivatized at a carbon or amino nitrogen or wherein the phosphate isreplaced with a phosphate analog (a “(ribonucleotide) phosphateanalog”). A ribonucleotide analog in a complex segment which is atemplate for the DDRP activity of an RNA replicase or in apolyribonucleotide made from such a complex segment on the basis of suchactivity is recognized by the replicase, in such a complex segment ofpolyribonucleotide, to place the ribonucleotide with the base, that iscomplementary to that of the analog, in the corresponding position ofthe autocatalytically replicatable RNA made from the complex segment orpolyribonucleotide. Among ribonucleotide analogs are rT and otherderivatives of U, wherein the uracil is derivatized at the 5-carbon(e.g., through a linker to biotin) and various phosphate analogscorresponding to phosphate analogs of the 2′-deoxyribonucleotides (seelisting above of 2′-deoxyribonucleotide phosphate analogs).

A 2′-deoxyribonucleotide, 2′-deoxyribonucleoside 5′-triphosphate(hereinafter referred to simply as a “2′-deoxyribonucleosidetriphosphate”), ribonucleotide or ribonucleoside triphosphate which ischanged only by changing the percentages of the various isotopes of anatom is not considered an “analog.”

The term “amplifiable probe” is intended to mean a nucleic acid whichhas the characteristics of a probe and has an amplifiable sequence.

In accordance with the terminology used in the present specification,two single-stranded nucleic acids will have the “same” sequence, even ifboth of them are chimeric, or one of them is an RNA and the other a DNAor chimeric, or one of them is a DNA and the other an RNA or chimeric,as long as both have the same number of nucleotides and the sequence ofbases on the nucleotides is the same in both. For purposes ofdetermining whether two nucleic acid sequences are the “same,” a basederivatized at one of its atoms (other than the nitrogen bonded to theribose) is considered to be the same as the underivatized base. Thus,for example, an RNA and a DNA will have the same sequence if they havethe same number of bases and the sequence of bases is the same in both,with each uracil in the RNA corresponding to a thymine in the DNA.

The term “silent sequence” or “silent segment” in reference toamplification is intended to mean a nucleic acid segment which, in afirst nucleic acid, is not amplifiable but which, in combination withother segment(s) in a second nucleic acid made with the first nucleicacid, is part of an amplifiable segment.

The term “Qβ replicase or its functional equivalent” as used herein isintended to mean an RNA replicase which catalyzes autocatalyticreplication of certain RNAs as well as, in accordance with the discoveryunderlying the present invention, synthesis of RNAs using as templatesDNAs and chimeric nucleic acids with the sequences of RNAs that areautocatalytically replicatahle by the replicase. For examples of suchreplicases, reference is made to PCT Patent Publication No. WO 88/10315to Gingeras, et al., PCT Patent Publication No. WO 87/06270 to Chu, etal., Blumenthal and Carmichael (1979) Ann. Rev. Biochem., 48:525-548,and Miyake et al., (1971) Proc. Natl. Acad. Sci. (USA) 68, 2022-2024, aswell as to references cited in these publications. Examples of suchreplicases that are useful in the present invention include the Qβreplicase, those encoded by the genomes of bacteriophages FI, f2, GA,MS2, R17, SD, SP, ST, VK, and ZR, as well as replicases of plant RNAviruses such as that of brome mosaic virus (BMV). Among thebacteriophage replicases, for example, there is interchangeability oftemplates for autocatalytic replication.

The skilled understand that there are many types of RNAs that areautocatalytically replicatable by Qβ replicase and other RNA replicases.Thus, among others, there are many so-called nanovariant RNAs and manyso-called midivariant RNAs. See, e.g., Chu, et al., PCT PatentPublication No. WO 87/06270 and references cited therein. DNAs, andother nucleic acid segments comprising a 2′-deoxyribonucleotide oranalog thereof, and corresponding to all of these RNAs are amplifiablevia the DDRP activity of RITA replicases. In the present application,nvDNA, unless otherwise qualified, refers to a specific DNA, namely thedouble-stranded DNA, one strand of which (referred to as nv(+)DNA) hasthe same sequence as the nanovariant(+) RNA (nv(+)RNA) taught bySchaffner, et al., 1977, supra, and the other strand of which, referredto as nv(−)DNA, has the exactly complementary sequence. See SEQ ID NO: 1and SEQ ID NO: 6 for the sequences of nv(+)DNA and nv(−)DNA,respectively. Examples of other nanovariant DNAs are given in Table 1 ofExample 3. Similarly, mdvDNA, unless otherwise qualified, refers to thedouble-stranded DNA with the sequence shown at SEQ ID NO: 23, one strandof which is referred to as mdv(+)DNA, because it has the sequence of amidivariant(+) RNA, and the other strand of which, with thecomplementary sequence, is referred to as mdv(−)DNA, because it has thesequence of the corresponding midivariant(−) RNA. MdvDNA is a“recombinant” midivariant DNA, as the corresponding RNA was made byinserting, using in vitro “gene-splicing” techniques with variousenzymes including restriction endonucleases, an RNA segment(corresponding to bases 66-75 in SEQ ID NO: 23) into a midivariant RNAthat is naturally occurring or arose in vitro in the course ofautocatalytic replication by Qβ replicase from a naturally occurring RNAtemplate for autocatalytic replication by that enzyme. See Kramer, etal., U.S. Pat. No. 4,786,600. “Nvplasmid” means a plasmid, such aspNV-1-3-4, which comprises nvDNA as a segment. “Nanovariant plasmid”means a plasmid which comprises a nanovariant DNA as a segment.“Mdvplasmid” means a plasmid, such as pMDV XhoI, which comprises mdvDNAas a segment. “Midivariant plasmid” means a plasinid which comprises amidivariant DNA as a segment.

The term “oligonucleotide primer” as used herein is intended to includeprimers, whether occurring naturally as in a purified restriction digestor produced synthetically, as with a DNA synthesizer, which are capableof priming DNA synthesis when placed under conditions in which synthesisof a primer extension product, which is complementary to a nucleic acidtemplate strand, is induced, i.e., in the presence of2′-deoxyribonucleotides or certain analogs thereof, as understood in theart, and an enzyme with DNA polymerase activity and at a suitabletemperature, pH and stringency for hybridization of primer to templateto occur and the enzyme to be active in catalyzing DNA synthesis. Theprimer is-provided in single-stranded form but may alternatively bedoubled-stranded. If double-stranded, the primer is first treated toseparate its strands before being hybridized to nucleic acid strandtemplate to initiate preparation of extension products. The primer mustbe sufficiently long to hybridize to template with sufficient stabilityto prime the synthesis of extension products in the presence of theenzyme providing DNA polymerase activity. A primer also may providespecificity by hybridizing specifically to a 3′-end of a target segment(or the complement thereof). The exact length of a primer will depend onmany factors, including temperature, stringency, and complexity of thetarget sequence. An oligonucleotide primer typically contains 20-35nucleotides, although it may contain fewer (down to about 6) or morenucleotides in the segment intended to hybridize with template. Shorterprimer molecules generally require lower stringency (e.g., coolertemperatures at constant pH, ionic strength, and otherstringency-determining factors) to form sufficiently stable hybridcomplexes with the template and tend therefore to be somewhat lessspecific with respect to segments to which they hybridize and initiatesynthesis.

Replicase Enzyme Activity

The present invention employs the use of RNA replicases, such as that ofRNA bacteriophage Qβ. The present invention employs a novel activity ofthe replicases, i.e., DNA-dependent RNA polymerase activity. Theactivity produces RNA copies from complex, DNA or chimeric nucleic acidsubstrates which have sequences of autocatalytically replicatable RNAsfor the replicase. The definition of “complex” with reference tosubstrates for RNA replicases is provided supra. The RNA copies providedby the DDRP activity are autocatalytically replicated by the samereplicase (or another that recognizes the RNA as a template forautocatalytic replication). The substrate for the Qβ replicase, forexample, can be any amplifiable DNA, e.g., nanovariant DNAs, midivariantDNAs, minivariant DNAS, other variants including those to which namesfor the corresponding autocatalytically replicatable RNAs have not beenassigned, or autocatalytically replicatable mutants thereof.

Prior to this invention, it had not been appreciated that RNA replicasesmanifest a DNA-dependent RNA polymerase activity capable of usingcomplex DNAs and chimeric nucleic acids as templates for making RNAs ofcomplementary sequence that are autocatalytically replicatable. Indeed,it has been reported that DNAs with complex sequences but havingterminal polydeoxycytidine were not active as templates forautocatalytic replication by Qβ replicase. Feix, G. and H. Sano (1976)FEBS Letters, 63:201-204.

Further, Feix and Sano, supra, reported that the DDRP activity theyobserved, narrowly limited with respect to template, was not increasedby the replacement of Mg⁺² with Mn⁺² in the reaction medium. However, inanother aspect of the present invention, it has been found surprisinglythat amplification of DNA and chimeric substrates, via the DDRP activityof RNA replicases, although apparently requiring Mg⁺², is enhanced inthe presence of divalent transition metal cations, such as Mn⁺², Co⁺²,or Zn⁺², in the reaction media at above about 0.5 mM, typically at nomore than about 5 mM and preferably at about 1 mM.

Reference herein to “about” with respect to a concentration or an amounthas the meaning ascribed to that term by practitioners in the molecularbiological and biochemical arts and, as such, generally means thespecified concentration or amount ±10%.

Templates

The DDRP activity of Qβ replicase and other RNA replicases is active onany complex DNA or chimeric nucleic acid segment which has the sequenceof an RNA that is autocatalytically replicatable by the replicase. Ithas been discovered, in connection with the present invention, that thereplicases are remarkably versatile in their capability to “identify” anucleic acid segment with a sequence of an RNA that is autocatalyticallyreplicatable with the replicase. Thus, such a complex DNA or chimericsegment can be free, in single-stranded form, with no nucleotides joinedto either of its ends. Alternatively, such a complex segment can besingle-stranded with nucleotides, which are not copied by the replicaseinto the RNA made with the DDRP activity, added at either or both ends.Still further, the complex segment can be all or part of one strand of adouble-stranded or partially double-stranded nucleic acid, including anhybrid nucleic acid, a nucleic acid in which the two strands are notexactly complementary in sequence, and a nucleic acid in which there maybe gaps (one or more nucleotides missing) or breaks (a severedphosphodiester bond but no nucleotides missing) in one or both strands.Indeed, a plurality of segments which, if covalently joined together,would form a complex segment that is a template for the DDRP activity ofan RNA replicase, will function as such a complex segment, even if notcovalently joined together, provided that the plurality of segments ishybridized immediately adjacent one another (i.e., with only breaks butno gaps between them) in the same order the segments would have in thecomplex segment, on a nucleic acid strand. Such a plurality of segmentsis referred to herein as a “broken complex segment.” The “sequence” of abroken complex segment is the sequence of the complex segment formed byclosing the breaks between the segments of the broken complex segment.Because the hybridization of the plurality of segments (preferably two)which constitute a broken complex segment needs to be sufficientlystable, each of the plurality of segments must have at least about 6,and typically at least about 10, bases in a segment complementary insequence to a segment of the other strand to which the plurality ofsegments hybridizes. The segment of the plurality at the 3′-end of thebroken complex segment and the segment of the plurality at the 5′-end ofthe broken complex segment need not be completely hybridized to theother strand; only a subsegment at the 5′-end of the segment of theplurality at the 3′-end and a subsegment at the 3′-end of the segment ofthe plurality at the 5′-end need be hybridized to the other strand. If,in the strand other than that with a first complex segment or brokencomplex segment, which is a template for autocatalytic replication by aRNA replicase, there is a second complex segment exactly complementaryin sequence to that of the first complex segment or broken complexsegment, the second complex segment will also be a template for DDRPactivity of the replicase. Whether single-stranded, double-stranded orpartially double-stranded, the nucleic acid in which a complex segment,with the sequence of an RNA that is autocatalytically replicatable by anRNA replicase, may be embedded and be operable as a template for theDDRP activity of the replicase can be in any physical form, linear(single-stranded or double-stranded), closed circular, super-coiled, orthe like. Thus, the complex DNA or complex segment that is a templatefor the DDRP activity of an RNA replicase can be a segment of a plasmid,including a relaxed or a super-coiled plasmid.

It has been discovered, then, in connection with the present invention,that a template in accordance with the invention for amplification withan RNA replicase can be provided to a sample as a pre-formed, singlenucleic acid, which comprises a complex segment which has the sequenceof an RNA that is autocatalytically replicatable by the replicase, or asone or more nucleic acids which can be processed or reacted in thesample to provide a nucleic acid which comprises a complex or brokencomplex nucleic acid segment which has the sequence of an RNA that isautocatalytically replicatable by the replicase.

The present invention provides a simple, straight-forward method foridentifying RNAS, including the many already known to the art, asdescribed above, which are templates for autocatalytic replication by Qβreplicase or other RNA replicases. Thus, employing methods well known tothe skilled, a DNA, most conveniently a plasmid or other vehiclesuitable for conveniently making significant amounts of DNA by cloning,is made which comprises a segment with the sequence of an RNA known tobe autocatalytically replicatable by an RNA replicase. As discovered inconnection with this invention, both strands of this segment will beamplifiable by the replicase, beginning with the DDRP activity of thereplicase. This segment can then be changed by any method known in theart, to delete, add, or change bases, and plasmid DNA with the changedsegment exposed to the replicase under conditions, such as thosedescribed herein, which will result in amplification if the DNA of themodified segment has the sequence of an RNA that. is autocatalyticallyreplicatable by the replicase. Detection of amplification can also becarried out as described herein.

For example, examples 3, 4, 5 and 8 below show segments with sequencesmodified form that of nv(+)RNA or nv(−)RNA which retain autocatalyticreplicatability by Qβ replicase. For example, with reference to Table 1of Example 3 and the Sequence Listing, SEQ ID NO: 9 differs fromnv(+)DNA SEQ ID NO: 1 by insertion of a 5′-GGAT between bases 15 and 16,insertion of a T between bases 45 and 46, change of base 48 from C to A,and insertion of a 24-base segment between bases 49 and 50. Similarly,again with reference to Table 1 of Example 3 and the Sequence Listing,SEQ ID NO: 10 differs from nv(+)DNA SEQ ID NO: 1 by the insertion of a48-base segment between bases 37 and 38. Similarly, mdvDNA could beeasily modified by, for example, insertion of DNA at the XhoI site toprovide additional DNAs that are amplifiable in accordance with theinvention.

Targets

Any sample containing a nucleic acid, in purified or nonpurified form,can be used to provide nucleic acid target, for the target-dependentprocesses of the invention, provided the sample is at least suspected ofcontaining the target. Examples include both DNA or RNA targets,including messenger RNAs, single-stranded or double-stranded RNAs orDNAs, or DNA-RNA hybrids. Further, the target nucleic acid segment maybe a small segment of a much larger molecule although it will typicallybe at least about 10 and more typically 20-50 nucleotides long. Furtherstill, the target nucleic acid may have a plurality of target nucleicacid segments, which may be the same or different.

Oligonucleotides

The present invention incorporates methods for the synthetic preparationof DNA, RNA or chimeric oligonucleotides. In this regard, reference ismade to Applied Biosystems Model 380B DNA Synthesizer Users Manual,Version 1.11, November, 1985; Beaucage, et al. (1981), TetrahedronLetts. 22:1859-1862; Mateucci and Carruthers (1981), J. Am. Chem. Soc.103:3185-3191; Sinka, et al Tetrahedron Lets. 24:5843-4846.

The oligonucleotides may be prepared using any suitable method, such as,for example, the phosphotriester and phosphodiester methods,phosphoramidite methods, or automated embodiments of any of them.

The present invention is directed to the use of the DNA-dependent RNApolymerase activity of Qβ replicase and other RNA replicases to generatemultiple reporter molecules. Each DNA or chimeric nucleic acid segment,which is a template for a replicase and is associated with a targetsegment can be used to generate greater than 10⁹ reporter molecules inthis fashion.

The present invention is also directed to several applications relatingto the discovery of the DDRP activity of RNA replicases. Examples offive different methods which have been devised to take advantage of thisdiscovery include the following. These various methods illustrate how acomplex segment or broken complex segment, which is amplifiable onaccount of the DDRP activity of an RNA replicase can be provided to.asample as a pre-formed, single nucleic acid, which comprises a complexsegment which has the sequence of an RNA that is autocatalyticallyreplicatable by the replicase, or as one or more nucleic acids which canbe processed or reacted in the sample to provide a nucleic acid whichcomprises a complex or broken complex nucleic acid segment which has thesequence of an RNA that is autocatalytically replicatable by thereplicase.

Example 1 Hybridization/Separation/Amplification

Example 2 Nuclease Protection/Amplification

Example 3 Ligation/Amplification

Example 4 Double Extension/Amplification

Example 5 cDNA Synthesis/Amplification

It will be explained in the following sections that several of thesemethods have more than one possible format depending on the number andcharacteristics of the probes being used.

In its most general sense, the invention is a method for amplificationof a nucleic acid molecule comprising at least one2′-deoxyribonucleotide and which amplification includes the use of theDDRP activity of an RNA replicase in one or more of its steps. Theinvention is also directed to methods for target nucleic acidsegment-dependent amplification of reporter molecules dependent uponsuch DDRP activity in one or more of its steps. Further, detection ofthese reporter molecules indicates presence of the target nucleic acidin a sample containing nucleic acid. The reporter molecules may haveuses other than to provide detectability of the presence of targetnucleic acid segment (and target nucleic acid) in a sample. These otheruses include use as probes, cloning intermediates, substrates forsequence analysis, and in other molecular biological or moleculargenetic methods.

The invention also entails kits for carrying out the methods.

These methods and kits are particularly usefully applied in connectionwith nucleic acid probe hybridization assays for detection of targetnucleic acid analytes. Thus, the invention also entails methods, andkits for carrying out the methods, for detecting the presence of anucleic acid analyte in a sample.

Again, the various aspects of the invention entail applications in (andrelated kits for) amplifying target segments, detecting target nucleicacid analyte, and other procedures, of the discovery that Qβ replicaseor another RNA replicase can use a complex DNA or chimeric nucleic acid,which has the sequence of an RNA that is autocatalytically replicatableby the replicase, as a template for catalyzing synthesis of the RNA ofcomplementary sequence. Because this RNA is also autocatalyticallyreplicatable by the replicase, the process of making the RNA from theDNA or hybrid nucleic acid initiates an autocatalytic replication of theRNA and its RNA complement catalyzed by the RNA-dependent RNA polymeraseactivity of the replicase. The substrate for the DDRP activity of areplicase in accordance with the invention must be a complex nucleicacid segment, as defined hereinabove, which has the sequence of a RNAthat is autocatalytically replicatable by the replicase.

Following are summaries of various of the many embodiments of thepresent invention.

Embodiment 1 Hybridization/Separation/Amplification

In this format, the method of amplifying a nucleic acid segment in asample includes mixing a probe with the sample containing the targetnucleic acid under hybridizing conditions. The free (i.e. unhybridized)probes are separated from those which are hybridized with the nucleicacid in the sample. The system with hybridized probes is then subjectedto amplification conditions and the amplified molecules are detected. Aprobe for this format may have the anti-target segment covalently linkedat either its 3′- or 5′- terminus to the replicase-amplifiable segmentor may have the anti-target segment embedded within the, and as part of,the replicase amplifiable segment. The reporter segment of the probe maybe the entire replicase amplifiable segment or a reporter subsegmentembedded within the replicase-amplifiable segment. The “reportersegment” has the sequence of the segment of amplified product that isassayed for in detecting whether amplification has occurred (i.e.,whether target nucleic acid is in the sample being analyzed.) The probemay be a linear molecule. Alternatively, the probe may be a circularmolecule wherein one terminus of the replicase-amplifiable segment isjoined directly (i.e., through a single phosphodiester) to theanti-target segment and the other terminus is joined to the anti-targetsegment either directly or through a connector segment.

With reference now to FIG. 1, the present invention in one aspect, is amethod for target nucleic acid segment-dependent amplification of areporter molecule, which method comprises the following steps 1a-1d asillustrated:

1a) An anti-target segment (sequence) covalently linked at either its 3′or 5′ terminus to an amplifiable sequence, e.g., nv(+)DNA, is mixed witha sample containing the target sequence under hybridizing conditions tocause the hybridization of the target and anti-target sequences.

1b) Column chromatography, or other means known to the art, is used toseparate the hybrids which have formed from the unhybridized probemolecules.

1c) The amplifiable segments of the hybrid molecules are amplified viathe DDRP activity of, e.g., Qβ replicase, to produce multiple RNAcopies, e.g., nvRNA.

1d) The amplified material generated in 1c may be detected by suitablemeans known in the art.

Reference is now made to FIG. 2, which schematically illustratesalternate probe constructs for use in thehybridization/separation/amplification format of FIG. 1. In the probesillustrated in the Figures, including FIG. 2, straight lines indicateentire, or partial, replicase-amplifiable segments (sequences); wavylines in probe indicate anti-target segments (which may also be part ofreplicase amplifiable segment) and wavy lines in target indicate targetsegments; the double line indicates a connector sequence; and the solidbox indicates a reporter segment. The terms “segment” and “sequence” areused interchangeably to mean a segment with a particular sequence.

In the process of FIG. 1, the probe molecule may have an amplifiablesequence joined directly (e.g., through a single phosphodiester) toanti-target sequence at either terminus (2a, 2d) or the anti-targetsegment may be internal to and part of the amplifiable segment (2b, 2c).A connector sequence may be used to circularize the probe (2c). Areporter segment may be present internal to the amplifiable segment(2d). Any of these probe constructs may be used in thehybridization/separation/ amplification format described above andillustrated in FIG. 1.

Embodiment 2 Nuclease Protection/Amplification

In this format, the probe comprises an anti-target segment adjacent the3′-end or the 5′-end of the replicase amplifiable segment. Theanti-target segment of the probe is selected, and the nucleic acid of asample thought to comprise target nucleic acid is treated, so that probehybridized to target is protected from digestion by a pre-selectednuclease. A sample of nucleic acid is hybridized with probe, thepre-selected nuclease is added to degrade probe that failed tohybridize, then amplification is effected by adding an RNA replicasewhich recognizes the replicase-amplifiable segment of the probe as atemplate for DDRP activity. The molecules made in the resultingamplification (if target was present) may be detected. Examples ofenzymes providing suitable nuclease activities include E. coliendonuclease VII, T4 DNA polymerase, and Klenow Fragment of E. coli DNApolymerase I.

Referring now to FIG. 3, there is schematically illustrated a method fora target nucleic acid segment-dependent amplification of a reportermolecule, comprising the following steps 3a-3d:

3a) In the nuclease protection/amplification format, a target nucleicacid sequence and a probe, comprising, attached directly at its3′-terminus, an amplifiable portion of nv(−)DNA, nv(+)DNA or otheramplifiable DNA is subjected to conditions which allow hybridization ofthe target and anti-target sequences to occur.

3b) The product of step 3a is subjected to nuclease digestion from the3′-terminus of unhybridized probe using the 3′- to 5′-single-strandednuclease activity of Klenow Fragment of E. coli DNA polymerase I, T4 DNApolymerase, or other suitable enzyme. The remaining probe molecules,protected from nuclease digestion because of their association withtarget via hybridization, can be amplified with a replicase tosynthesize amplification products (i.e., reporter molecules).

3c) The strands of probe which survived the nuclease digestion step inaccordance with Step 3b are amplified using Qβ replicase, or anotherreplicase, and relying on the DDRP activity of such enzyme using astemplate the amplifiable segment of the probe.

3d) The molecules generated in accordance with the amplification of Step3c may be detected by suitable means known to those skilled in the art.

Embodiment 3 Ligation/Amplification

In this format, the sample is treated under hybridizing conditions witha first non-amplifiable probe and a second non-amplifiable probe, eachprobe comprising part of an amplifiable nucleic acid segment, said partjoined directly to anti-target nucleic acid sequences. In one probe, theanti-target sequence is joined at its 5′end to a 5′-part of anamplifiable segment. In the other probe, the anti-target sequence isjoined at its 3′-end to the 3′-remainder of the amplifiable segment. Theanti-target sequences are selected so that, when hybridized to target,they will be adjacent one another and are capable of being ligated.After hybridization, the first and second probes are joined by treatmentwith a ligase enzyme, such as T4 DNA ligase or E. coli DNA ligase, toproduce a replicase-amplifiable molecule. Upon amplification, theamplified molecules may then be detected.

Referring now to FIG. 4, there is schematically illustrated a method,involving the Ligation/Amplification format, for a target nucleic acidsegment-dependent amplification of a reporter molecule, comprising thefollowing steps 4a-4d.

4a) Two non-amplifiable probes, A and B, each contain a part of anamplifiable sequence (both parts together being the amplifiablesequence), e.g., nv_(A) and nv_(B), respectively, linked directly toportions of an anti-target sequence, e.g., anti-target A and anti-targetB respectively. The two probes are mixed with a nucleic acid comprisingtarget under hybridizing conditions.

4b) The probes are ligated via the anti-target sequences using T4 DNAligase, E. coli DNA ligase or other enzyme to provide suitable ligaseactivity to produce a molecule which is amplifiable.

4c) The ligated probes are amplified using a replicase (e.g. Qβ) andrelying on its DDRP activity.

4d) The amplified material generated in accordance with Step 4c may bedetected by suitable means known to those skilled in the art.

Several modifications of this format may be employed. First, it ispossible to use as probe a single molecule, wherein the 5′-terminus ofthe 5′probe (probe A in FIG. 4) is joined directly (e.g., by aphosphodiester bond or other short covalent linkage that does not entaila nucleoside) to the 3′-terminus of the 3′-probe (probe B in FIG. 4).Alternatively, the probe may be circular, i.e., the termini may bejoined by a connector sequence, or a circle may be formed by theligation after hybridization. The ligation would then result in asingle-stranded circle with an amplifiable segment.

A second modification is to eliminate the ligation step and employ thebroken complex segment as the template for the DDRP activity. Althoughthe efficiency of amplification is reduced by this alteration, a step inthe procedure is saved.

A third modification of this method is to design the two probes suchthat anti-target A and anti-target B hybridize to sequences which arenot precisely adjacent to one another. In this case, an additional DNApolymerization step which follows hybridization and precedes ligation,will fill in the intervening sequence to form a broken complex segment,which may be used as a template for the DDRP activity without ligationor may be ligated and then used as a template for the DDRP activity.This altered format offers the advantage of amplification of thesegments (the sequences of which might not be known) between the targetsegments in addition to the amplification of target (and anti-target)segments.

Finally, one or both probes may be amplifiable by themselves. In suchcases, the amplified products of ligated molecules will differ fromthose of the probes alone. This difference may be detected using generalmethods of analysis known to those skilled in the art.

Embodiment 4 Double Extension/Amplification

In this format, a probe consisting of a portion (including the5′-terminus) of a replicase-amplifiable sequence covalently joined atits 3′-terminus with an anti-target sequence is mixed under hybridizingconditions with a sample containing a nucleic acid. The hybridsresulting if target is present are treated with an enzyme providing DNApolymerase or reverse transcriptase activity to extend the hybridizedprobe from the 3′-terminus in a primer-dependent extension reactionusing target as template. The product of the extension is separated fromthe target, as by thermal denaturation, and hybridized with a secondprobe consisting of a portion (including the 5′-terminus) ofreplicase-amplifiable sequence covalently joined at its 3′ terminus withsequence that is the same as that of a sequence of target that islocated 5′ from the target sequence of the first probe. The portion ofthe amplifiable sequence of the second probe is from an amplifiablesequence that is the complement of the amplifiable sequence, of which aportion is at the 5′-terminus of the first probe. The sequence of targetin the second probe is complementary to a sequence in the part ofextended first probe added in the extension. Thus, hybridization of thesecond probe will occur with the extended product of the first probe,but not with the first probe itself. The denatured extended first probeis hybridized with the second probe. The resulting hybrid is used as atemplate for a primer extension by an enzyme providing DNA polymeraseactivity. The product of this extension is amplified with a replicase,and the amplified molecules are detected.

Examples of enzymes providing the DNA polymerase activity that may beused in the primer extensions, of this or any other embodiment of theinvention, are E. coli DNA polymerase I, Klenow Fragment of E. coli DNApolymerase I, avian myeloblastosis virus reverse transcriptase, Moloneymurine leukemia virus reverse transcriptase, Thermus aquaticus DNApolymerase, M. luteus DNA polymerase, T4 DNA polymerase, T7 DNApolymerase, Thermus thermophilus DNA polymerase, Thermus flavus DNApolymerase, Bacillus licheniformis DNA polymerase, Bacillusstearothermophilus DNA polymerase, or other DNA polymerases, reversetranscriptases, or enzymes with a primer-initiated, template-dependentDNA polymerase activity. Examples of enzymes providing the reversetranscriptase activity that may be used in the primer extensions, ofthis or any other embodiment of the invention, are avian myeloblastosisvirus reverse transcriptase, Moloney murine leukemia virus reversetranscriptase, the reverse transcriptase of any other retrovirus or of aretrotransposon, Thermus aquaticus DNA polymerase, or other enzymes withreverse transcriptase activity.

The two probes may be added at different times or simultaneously,although if the strand-separation of the first extension product fromtarget employs thermal denaturation at a temperature that denatures thepolymerase employed in the first extension, additional polymerase willneed to be added for the second extension.

Referring now to FIG. 5, this embodiment relates to a method for atarget nucleic acid segment-dependent amplification of a reportermolecule using a Double Extension/Amplification format and comprises thefollowing steps 5a-5f:

5a) Two probes A and B, as described for the DoubleExtension/Amplification Format, are employed. These probes are notcomplementary to one another, i.e., prior to the extension of Probe A,the two probes are not capable of hybridizing to one another underhybridizing conditions employed in the procedure with sufficientstability to prime a template-dependent, primer-initiated DNA extensionreaction). Probe A may comprise at its 5′-end a non-amplifiable,5-′-portion of nv(+)DNA. Probe B would then comprise at its 5′-end anon-amplifiable 5′-part of nv(−)DNA (i.e., the nanovariant DNA strandwith the sequence complementary to that of the nanovariant DNA strand ofwhich the 5′-end of Probe A is a part). The anti-target sequences of thetwo probes will provide specificity and be effective to primeprimer-dependent DNA synthesis on templates; thus, they will be at leastabout 10 and more typically 20-50 nucleotides in length. In step 5a, themixture of Probe A and nucleic acid is subjected to conditions whichcause the hybridization of target with anti-target sequences in theProbe.

5b) The hybrid which occurs if target is present is treated with a DNApolymerase or reverse transcriptase to generate adjacent anti-targetnucleic acid sequences by primer extension from the 3′-terminus of ProbeA as primer hybridized in accordance with Step 5a.

5c) The extended probe strands produced in accordance with Step 5b areseparated from the original target by thermal denaturation. The originalprobe A, now having an extended sequence as created in accordance withStep 5b, is then hybridized with probe B. Depending on the segment oftarget nucleic acid selected to provide the sequence of the3′-anti-extended-Probe A segment of Probe B, Probe B may hybridizeimmediately adjacent to the anti-target segment originally present inprobe A, or to a segment of extended Probe A that is 3from the 3′-end ofthis “original” anti-target segment. Typically the segment to whichprobe B hybridizes will be within 2000 nucleotides of the 3′-end of theoriginal anti-target segment and usually much closer. It is noteworthythat carrying out this method does not require knowledge of the sequenceof the segment of target between the 3′-end of the target segment ofprobe A and the 5′-end of the target segment also present at the 5′-endof probe B.

5d) An amplifiable DNA is then generated by primer extension from ProbeB as primer hybridized to extended Probe A in accordance with step 5c.

5e) The amplifiable molecule(s) generated in Step 5d is amplified via areplicase, employing its DDRP activity.

5f) The amplified material may be detected by suitable means known tothose skilled in the art.

The process may be modified by using, as Probe A or Probe B or both,probe(s) that is (are) amplifiable. In this case, the amplified materialproduced in Step 5e can be distinguished from that of the originalprobes A (and B) by suitable means known to those skilled in the art.Additionally, the process may be modified to include other uses ofamplified material in addition to providing detectability for thepresence of target in a sample.

Embodiment 5 cDNA Synthesis/Amplification

In this format, an RNA probe consisting of a replicase-amplifiablesequence covalently joined at its 3′-terminus with an anti-targetsequence is mixed with the nucleic acids in the sample under hybridizingcondition. The hybridized molecules are then treated with a reversetranscriptase enzyme. The RNA portion of the resulting RNA-DNA hybrid,and unhybridized RNA probe, are then destroyed. All or part of theremaining DNA sequence is then amplified using a replicase enzyme, thatcan be employed to amplify the replicase amplifiable segment of the RNAprobe, and the resulting amplified molecules may then be detected.Examples of the reverse transcriptase enzyme include avianmyeloblastosis virus reverse transcriptase, Moloney murine leukemiavirus reverse transcriptase, and Thermus aquaticus DNA polymerase. Thedestruction of unhybridized probe and the RNA portion of the RNA-DNAhybrid is enhanced under basic conditions, such as by the addition ofsodium hydroxide. The destruction of the RNA portion of the RNA-DNAhybrid may also be accomplished enzymatically with suitable enzymes,such as RNase H from E. coli or other species. Free probe can bedigested using various ribonucleases.

Referring now to FIG. 6, this embodiment is a method for a targetnucleic acid segment-dependent amplification of a reporter molecule,employing the CDNA Synthesis/Amplification Format and comprising thefollowing steps 6a-6e.

6a) A target nucleic acid sequence is hybridized with an amplifiable RNAprobe comprising a portion of nv(+)RNA, nv(−)RNA or other amplifiableRNA attached covalently to anti-target nucleic acid sequence(s) at its3′ terminus. The 3′-end of the target segment is at the 3′-end of targetmolecule and, in the hybrid with RNA probe, is complementary to thenucleotide at the 5′-end of the anti-target segment.

6b) The strands of the hybrid molecule are elongated by primer extensionin the presence of AMV Reverse Transcriptase or another suitable reversetranscriptase.

6c) Unhybridized RNA probe and chain-extended RNA is digested eitherchemically, e.g., and sodium hydroxide treatment or enzymatically, e.g.,RNase treatment. Unhybridized probe may be removed prior to step 6b.

6d) The treated sample is neutralized with an acid or buffer (in thecase of sodium hydroxide treatment described in Step c) or RNaseinhibitor (in the case of RNase treatment).

6e) The DNAs generated in Step c have amplifiable segments, and theseare amplified via the DDRP activity of a replicase which is capable ofautocatalytically replicating the amplifiable segment of the RNA probe.

The amplified material may be detected by suitable means known to thoseskilled in the art.

Several specific additions and modifications to this format may beuseful for specific applications. For example, the method requires thata 3′ terminus terminal hydroxyl be available at the end of the targetsequence for the elongation process described in Step 6b. If the targetdoes not present itself in this fashion, digestion of the targetsequence at a defined site with a restriction endonuclease prior todenaturation and hybridization is one option. A second option is togenerate random target 3′-ends by shearing, chemical cleavage, ordigestion with nucleases prior to hybridization. A third option is totreat the hybrids formed in Step 6a in the presence of both an enzyme toprovide 3′- to 5′-exonuclease activity and an enzyme to provide reversetranscriptase activity. The exonuclease activity will trim back theoverhanging 3′-terminus of the hybridized sample nucleic acid to theportion which complements the anti-target portion of the probe. Thereverse transcriptase activity will then extend the sequence from thehybridized 3′-hydroxyl terminus.

Referring now to FIG. 7, a fourth method for generation of the required3′-terminal hydroxyl at the end of the target sequence comprises thefollowing steps 7a-7d:

7a) A target nucleic acid is hybridized with two probes, A and B, suchthat, when hybridized, the probes are separated by an intervening gap ofat least one, and more typically at least several, up to about 2000,nucleotides. Probe A may be DNA, RNA or a chimeric nucleic acid. Probe Bis preferably DNA as it and its extension products must be resistant todegradation under conditions which degrade RNA.

7b) Probe B, which is hybridized to a target segment located 3′ from thesegment to which Probe A hybridizes, is elongated by primer extension inthe presence of T7 DNA polymerase, T4 DNA polymerase, E. coli DNApolymerase I, or Klenow Fragment thereof, or another suitable polymeraseor reverse transcriptase to catalyze the extension reaction.

7c) The extended probe B is separated from the target nucleic acid bymeans familiar to those skilled in the art, e.g. thermal denaturation.Note that the extension of Probe B is blocked by Probe A to provide adefined, 3′-end to Probe B.

7d)-7h) The separated, extended probe B is then used, with a thirdprobe, probe C, which is an RNA with the same functional properties,relative to extended probe B, as the RNA probe of FIG. 6 to generatereporter molecules which may be detected. Steps 7d)-h) correspond tosteps 6a)-6e), respectively.

Another modification of the cDNA Synthesis/Amplification format is toreplace the RNA probe with a chimeric molecule comprisingdeoxyribonucleotides and at least two ribonucleotides, such that thereplicase-catalyzed autocatalytic replicability of the chimeric moleculecan be destroyed with alkaline or RNase treatment. PM1500, which has twopairs of ribonucleotides, is an example of an RNA that could be used asthe amplifiable segment of such a chimeric probe, albeit the processusing a probe with such an amplifiable segment proceeds withsubstantially reduced efficiency in comparison with the completely RNAprobe of the same sequence, as complete digestion of the chimeric probewhich is required to reduce “background” to a minimum is more difficultthan with the completely RNA probe. All the steps of the method with achimeric in place of a completely RNA probe can be performed asdescribed in this section.

Detection Methods

The detection of amplified products can be performed by methods andmaterials familiar to those skilled in the art. Such detection methodsinclude reactions of RNA with dyes and detection of the dye-RNAcomplexes. Especially in situations where the RNA amplification productis present in a significant background of other nucleic acids, whichwould also form complexes with a dye, detection of amplification productby formation of dye-RNA complexes can be accompanied by separation (asby electrophoresis, chromatography or the like) according to size ofnucleic acid of a sample thereof in which an amplification reaction hasbeen carried out in order to detect the product(s) of the amplificationreaction, which will have characteristic size(s). Confirmation thatnucleic acid of the expected size found in a sample using dye-stainingafter an amplification reaction according to the invention is RNA fromthe amplification reaction can be obtained by using a sequence-specificdetection method, such as a nucleic acid probe hybridization method, asdescribed below. The dyes include chromogenic dyes such as “stains all”(Dahlberg, et al. (1969), J. Mol. Biol., Vol 41, pp. 139-147), methyleneblue (Dingman and Peacock (1968), Biochemistry, Vol. 7, pp. 659-668) andsilver stain (Sammons, et al. (1981), Electrophoresis, Vol. 2, pp.135-141; Igloi (1983), Anal. Biochem., Vol. 134, pp. 184-188) andfluorogenic compounds that bind to RNA, including ethidium bromide(Sharp, et al. (1973), Biochemistry, Vol. 12, pp. 3055-3063; Bailey andDavidson (1976), Anal. Biochem., Vol. 70, pp. 75-85), acridine orange,propidium iodide and ethidium heterodimer.

Additional means of detection which are familiar to those skilled in theart include the use of modified ribonucleoside triphosphates during theamplification reaction, leading to incorporation of modified, detectableribonucleotides specifically into the amplified products, followed byseparation (e.g., chromatographically, electrophoretically) ofamplification products from unincorporated, modified ribonucleosidetriphosphates, prior to detection of the amplified products based onsignal directly from the label of the modified, incorporatedribonucleotides or produced by subsequent reactions of the amplifiedproducts dependent on the presence of such label. Most commonly, amodified ribonucleotide is radioactively labeled with an isotope such as³²P or ³⁵S. The detection of beta particle emissions from such isotopesincorporated into RNA resulting from amplification according to theinvention is performed by methods, such as scintillation counting orautoradiography, well known in the art. Ribonucleoside triphosphates,which are modified to carry a luminescent, fluorescent or chromogenicmoiety on the base, can also be incorporated into the amplificationproduct and then detected by various methods and means familiar to thoseskilled in the art. Other modifications of ribonucleoside triphosphatesthat can be tolerated by the replicases for incorporation of themodified ribonucleotides into amplification products include those wherethe bases are linked to “affinity molecules” such as biotin, antigens,enzyme inhibitors, or the like, which provide detectability to theamplification products through subsequent reaction with, e.g.,enzyme-labeled avidin or streptavidin reactive with biotin,enzyme-labeled antibody specific for an antigen affinity molecule, orcomplex of enzymes reactive with an enzyme-inhibitor affinity molecule,as understood by the skilled. For example, reaction of biotin linked toan uracil moiety in amplification product with avidin or streptavidinconjugated to a detectable material as described previously or to anenzyme to catalyze a reaction with substrates which react to producedetectable (e.g., colored) materials, is a familiar means of detectionto those skilled in the art.

Additional means of detection of products of amplification in accordancewith the invention which are familiar to those skilled in the artinclude hybridization of a sample of nucleic acid thought to includesuch product with a nucleic acid probe which comprises a segment withthe entire sequence of the product or a pre-selected portion of suchsequence (a “reporter” sequence or segment). Note that the amplificationproduct, because it results from a process including autocatalyticreplication, will include RNA with the sequence of the DNA segment thatwas the substrate for the DDRP activity of the replicase and RNA withthe complementary sequence. Probes to both such RNAs may be employedsimultaneously, particularly in situations where one of the two might bepresent in a significant excess over the other. The nucleic acid probewill be labelled in some way to make it detectable, e.g., will includeat least one radioactively labelled or otherwise modified nucleotide asdescribed above in connection with labelling of the amplificationproduct per se or may be labelled directly (covalently and prior to usein hybridization with target of the probe), with an enzyme which cancatalyze a signal-producing (e.g., chromogenic) reaction. Methods andmeans for detecting amplification product via nucleic acid probehybridization are also well known to the art. For example, ifnucleotides which carry biotin are incorporated into the nucleic acid asdescribed in Forster (1985), Nucleic Acids Res. and Lange (1981), Proc.Natl. Acad. Sci., USA, the products may be detected by first reactingthem with a conjugate of avidin or streptavidin with a signalling moietyand then by detection of the signalling moiety. The signalling moietiescould include luminescent, fluorescent or colored (chromogenic)compounds, enzymes which convert reactants to one of such compounds, oranalytes which react in the presence of other reactants and/or an enzymeto produce a luminescent, fluorescent, or colored compound.

When the amount of the reporter RNA produced in an amplificationreaction according to the invention is substantial, as the skilledunderstand, both in absolute amount (so that when complexed with a dyethe RNA would be detectable even if no other nucleic acid were present)and compared with the amount of the nucleic acid of the original sample(so that “background” due to complexes between the dye and other nucleicacid will not make the complex of the dye with reporter RNAundetectable), the incorporation of radioactively or otherwise modifiedribonucleotides or analysis by nucleic acid probe hybridization assaymethods is not required for detection of amplified product. Theamplified material can be detected directly by reaction withluminescent, fluorescent or colored dyes, often after separationaccording to size from other nucleic acids by, e.g., gelelectrophoresis. The person of ordinary skill is capable of determiningreadily, for a given dye, given size of RNA product from amplification,and given process used for separation of nucleic acid by size, what theminimum detectable amount of amplification product would be if no othernucleic acid were present. Generally, amplification in accordance withthe invention to provide RNA, made by autocatalytic replication, in atleast a 100-fold molar excess relative to the DNA or chimeric templatefor DDRP activity with which the amplification is initiated will beadequate to distinguish such RNA, complexed with dye, from other,similarly sized nucleic acids in a sample, provided that the 100-foldmolar excess of RNA is above the minimum detectable amount in the systemthat is used. As the skilled will understand, in many situationsamplification to much lower levels (e.g., to provide only a 5-fold molarexcess) will be adequate to provide detectability above background. Inany case, amplifications to provide far in excess of the above-noted100-fold molar excess will typically be carried out.

Alternative detection methods include detection of accumulation ordepletion of one of the reagents involved in the amplification process.For example, during autocatalytic replication, the ribonucleosidetriphosphate ATP is consumed as AMP is incorporated into the reporterRNA molecules. The concentration of ATP can be measured accurately usingknown methods which rely on bioluminescence catalyzed with a luciferase,such as a beetle luciferase (e.g., from P. pyralis). Thus, amplificationin accordance with the invention could be detected by usingbioluminescence catalyzed by a luciferase to detect depletion of ATPfrom a solution in which such amplification was occurring.

General Separation Methods After Amplification

The separation of RNA produced in amplification by autocatalyticreplication and containing either normal or modified nucleotides orbound with dyes is generally conducted by methods and means known to theart. For example, amplified materials can be bound to filters orparticles and unbound modified nucleotides or dyes can be separated andremoved by suitable washing conditions. The binding process can benon-specific, e.g., binding all nucleic acids, but not unincorporatedmaterials; or specific, binding only nucleic acids comprising particularsequences. or other properties. Specific binding can be directed bysubstances that are bound to any of various support materials (e.g.,surfaces of wells on microtiter plates, latex or agarose beads(including magnetic beads), chromatographic resins, as understood in theart) and that are capable of complexing specifically with certainnucleic acids. For example, when the nucleic acid to be specificallybound is RNA amplification product resulting from amplification inaccordance with the invention, such specific-binding substances includeantibodies to specific classes of nucleic acids, e.g., double-strandedRNA, nucleic acids comprising a segment with a specific sequencecomplementary to a sequence in amplified product; or avidin orstreptavidin to complex with biotin in the RNA produced in theamplification process as described previously.

Applications of the Invention Other than Production of ReporterMolecules

The products resulting from the DDRP activity of Qβ replicase and otherRNA replicases may be used as nucleic acid probes in essentially anyapplication in which RNA probes can be used. For example, a nanovariantRNA in which a probe sequence is incorporated can be made starting witha nanovariant DNA segment of the same sequence (or complementary) of thenanovariant, probe-sequence-containing RNA using the DDRP activity of areplicase and can be used in several hybridization formats which involvesolid supports, including Southern hybridization, Northernhybridization, slot blot and dot blot hybridization, and in situhybridization. Hybridization on other solid surfaces, such as latexbeads or paramagnetic particles, and in solution may also be effectiveuses of the molecules from amplification. The RNA probe may, asindicated above, be labelled in the process of being made from the DNAor, being autocatalytically replicatable, can be used unlabelled tohybridize with target that may be present in a sample of nucleic acidsbeing probed and then, if such hybridization has occurred, can besubjected to conditions for further autocatalytic replication (possiblywith simultaneous labelling as described above) prior to detection.

The products from amplification in accordance with the invention couldalso be used in gene expression work. Introduction into an amplifiableDNA sequence of a cassette containing a translation initiation siteupstream of a sequence coding for a peptide or protein, and use of theRNA, made by autocatalytic replication begun with the DDRP activity of areplicase using this construct as a template, in combination with atranslation system, such as an X. laevis oocyte system or an in vitrorabbit reticulocyte lysate system, could yield significant amounts of aprotein of interest.

The products from amplification could also be used as substrates forsequence analysis using standard methods for sequencing of RNA familiarto those skilled in the art.

In accordance with the present invention, complex amplifiable DNAs orchimeric nucleic acids could also be used in place of autocatalyticallyreplicatable RNAs to label “affinity molecules,” including antibodies,nucleic acid probes, and the like, as described, e.g., in Chu et al.,PCT Application Publication No. WO87/06270 or U.S. Pat. No. 4,957,858,used in detecting analytes to which the affinity molecules bindspecifically. The amplifiable, complex DNA or chimeric nucleic acidlabel could be treated, substantially as described in PCT ApplicationPublication No. WO87/06270 or U.S. Pat. No. 4,957,858 forautocatalytically replicatable RNA label, to provide detectability to anaffinity molecule. The RNA affinity molecules described in PCTApplication Publication No. WO87/06270 and U.S. Pat. No. 4,957,858,which are autocatalytically replicatable by Qβ replicase or another RNAreplicase and which also comprise an anti-target segment correspondingto a nucleic acid analyte can, in accordance with the present invention,be replaced with DNAs or chimeric nucleic acids of the same sequence or,as described above, readily made from such DNAs or chimeric nucleicacids. U.S. Pat. No. 4,957,858 is incorporated herein by reference.

Kits

In other embodiments, the invention relates to kits for carrying out thetarget nucleic acid segment-dependent amplification of reportermolecules according to the methods described above and to diagnostickits for the detection of specific target nucleic acid analytes in asample containing one or more nucleic acids in which at least one of thenucleic acids is suspected of containing a pre-selected target sequence.The kits are preferably packaged in multicontainer units havingindividual containers for each component. Examples of kits relating tothis invention are as follows:

EXAMPLE 1 (Kit 1) Hybridization/Separation/Amplification Kit

The Hybridization/Separation/Amplification Kit comprises at least twocontainers, packaged together, with the following components in separatecontainers:

(a) a hybridization solution comprising an oligonucleotide probe havinga complex, amplifiable nucleic acid segment or a portion thereof and ananti-target nucleic acid segment (see the description below of kit 3 forthe case that the probe has only a portion of an amplifiable segment, insuch a case there must be at least two probes); and

(b) an amplification buffer with Qβ replicase or another RNA replicase,which has DDRP activity with the amplifiable segment of the probe ofcomponent (a), said buffer suitable for the DDRP activity of saidreplicase.

A preferred hybridization solution comprises the following: 5×SSC (750mM NaCl, 75 mM sodium citrate), 2% dextran sulfate, 40 mM sodiumphosphate, pH 6.5, 0.1 mg/ml sheared and denatured herring sperm DNA,0.02% ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin(Pentax Fraction V).

A preferred amplification buffer for Qβ replicase comprises thefollowing: 40 mM Tris·HCl, pH 7.5, 10 mM MgCl₂, and 1 mM each of rATP,rGTP, UTP, and rCTP.

A hybridization/separation/amplification kit may also include buffersand other components (e.g., columns with gel) to carry out separation ofhybridized from unhybridized probe.

EXAMPLE 2 (Kit 2) Nuclease Protection/Amplification Kit

The Nuclease Protection/Amplification kit comprises at least threecontainers, packaged together, with the following components in separatecontainers:

(a) a hybridization buffer comprising an oligonucleotide probe with acomplex amplifiable segment and the other properties described above foruse in accordance with the nuclease protection/amplification method;

(b) an exonuclease buffer containing an exonuclease to catalyzedegradation, in accordance with the nuclease protection/amplificationmethod, of any probe that does not hybridize with target in an assay;and

(c) an amplification buffer as in Kit 1.

A preferred hybridization buffer and amplification buffer are describedabove in the description of Kit 1.

A suitable exonuclease buffer comprises the following: 40 mM Tris·HCl,pH 7.5, 10 mM MgSO₄, and 0.1 mM dithiothreitol.

EXAMPLE 3 (Kit 3) Ligation/Amplification Kit

The Ligation/Amplification Kit comprises, packaged together, at leasttwo containers containing, in separate containers, the followingcomponents:

(a) a hybridization solution with the oligonucleotide probes, at leastone of which is DNA or chimeric, which ligated together would comprise acomplex amplifiable segment, and which have the other propertiesdescribed supra for probes employed in accordance with theligation/amplification methods of the invention; and

(b) a ligase/amplification buffer containing T4 DNA ligase or otherligase and Qβ replicase or other RNA replicase (capable of amplifyingvia DDRP activity the amplifiable segment(s) occurring in the probes ofcomponent (a) when hybridized adjacent one another), said buffer beingsuitable for ligation with the ligase of single-stranded breaks in onestrand of a double-stranded DNA and for DDRP activity of the replicase.

A preferred hybridization solution is described above in the descriptionof Kit 1.

A preferred ligase/amplification buffer comprises the following: all ofthe components of the amplification buffer of Kit 1, plus 1 mM ATP, and0.05 mg/ml bovine serum albumin.

Note that the amplification buffer of Kit 1 could be employed in placeof the ligation/amplification buffer if probes hybridized to target arenot to be ligated.

EXAMPLE 4 (Kit 4) Double Extension/Amplification Kit

The Double Extension/Amplification Kit comprises, packaged together, atleast three containers, with each of the following components inseparate containers:

(a) a hybridization solution with the oligonucleotide probes, with theproperties described hereinabove for the extension/amplification method,to yield a complex nucleic acid amplifiable via the DDRP activity of anRNA replicase;

(b) an extension buffer containing a DNA polymerase or reversetranscriptase to provide DNA polymerase activity; and

(c) an amplification buffer as in Kit 1.

A preferred hybridization solution is described above in the descriptionof Kit 1. A preferred extension buffer comprises the following: 40 mMTris·HCl, pH 7.5, 10 mM MgSO₄, 0.1 mM dithiothreitol, and 0.04 mM eachof dATP, dCTP, dGTP and TTP.

EXAMPLE 5 (Kit 5) cDNA Synthesis/Amplification Kit

The cDNA Synthesis/Amplification Kit comprises, packaged together, atleast four containers with each of the following components in separatecontainers:

(a) a hybridization solution with an RNA- orchimeric-amplifiable-segment containing oligonucleotide probe, asdescribed hereinabove for the cDNA synthesis/amplification method of theinvention (see procedure 5, above) and the other probes that may beemployed in embodiments of the method (see procedure 6 above);

(b) a reverse transcriptase buffer containing AMV reverse transcriptase,MMLV reverse transcriptase, or other reverse transcriptase, said bufferbeing suitable for catalysis of reverse transcription by the enzyme;

(c) a solution to degrade RNA or chimeric probe after reversetranscription primed by target on the probe; and

(d) an amplification buffer as in Kit 1 with a replicase (e.g., Qβreplicase) capable of amplifying the amplifiable segment of the RNA orchimeric probe in component (a).

A preferred hybridization solution and amplification buffer aredescribed above in the description of Kit 1. A preferred reversetranscriptase buffer comprises the following: 34 mM Tris·HCL, pH 8.3, 50mM NaCl, 5 mM MgCl₂, 5 mM dithiothreitol, and 1 mM each of dATP, dGTP,TTP, and dCTP. A preferred RNA-degrading solution is 1 N NaOH.

EXAMPLE 6 (Kit 6) Hybridization/Amplification Kit

The Hybridization/Amplification Kit is the same as theHybridization/Separation/Amplification Kit (Kit 1) described above butincludes no components for separation of hybridized from unhybridizedprobe.

Kits of the invention may also include reagents for detection, or otheruses, of RNA produced in amplification in accordance with the invention.

As the skilled understand, diagnostic assays and other tests such asthose contemplated in connection with the present invention aregenerally carried out on test sample(s) in parallel with suitablepositive or negative “control” samples to insure that the reagentsemployed in the assays or tests are functioning properly to generate asignal indicative of the presence of an analyte, if analyte is presentin a test sample, and to provide a level of “background” signal(typically signal from a control sample known to have none of theanalyte), which signal obtained from a test sample must exceed before itcan be concluded reliably that the test sample included analyte. Controlsamples can also be employed to provide a measure of signal as afunction of the amount or concentration of analyte and, thereby, allowquantitation of the amount or concentration of analyte in test samples.Further, “control” analytes known to be present in test samples, in somecases at known concentrations (e.g., two beta-hemoglobin genes pernormal red blood cell), or deliberately added to test samples, alsopossibly at known concentrations, can be employed to provide suitablecontrols or standards for quantitation in testing for analytes beingtested for in test samples. The kits according to the invention,especially the test kits for analytes, may also include probes and otherreagents to provide suitable controls for the use of the kits indetermining the presence of or quantifying the amount of analytes intest samples.

The following examples are offered by way of illustration and are notintended to limit the invention in any manner.

EXAMPLES Example 1

This is an example of amplification of a DNA employing the DDRP activityof an RNA replicase, Qβ replicase. The amplified DNA is a nanovariantDNA with the sequence specified in SEQ ID NO: 1, namely 5′-GGGGAAATCCTGTTACCAGG ATAACGGGGT TTTCTCACCT CTCTACTCGA AAGTTAGAGA GGACACACCCGGATCTAGCC GGGTCAACCC-3′. The nanovariant DNA with this sequence isreferred to in the present specification as “nv(+)DNA”. The nanovariantDNA with the sequence complementary to that of nv(+)DNA is referred toin the present specification as “nv(−)DNA”. The 90-base pairdouble-stranded DNA, one strand of which is nv(+)DNA and the otherstrand of which is nv(−)DNA, is referred to in the present specificationas “nvDNA”. A 50 attomole sample of nv(+)DNA was amplified with thefollowing steps: The 50 attomoles of nv(+)DNA was taken up in 10 μlfinal volume of a mixture containing the following:

10 M Tris·HCl , pH 7.5;

15 mM MgCl₂

1 mM each of the ribonucleoside

triphosphates ATP, GTP, UTP, CTP; and

100 μg/ml Qβ replicase.

The mixture was incubated at 30° C. for 60 minutes, and transferred to amicrotiter well containing 10 μl of a 2×concentrate of reaction stopsolution (2×reaction stop solution: 100 mM EDTA, 0.2% NaPP_(i) (i.e.,sodium pyrophosphate), 1 μg/ml ethidium bromide). The reaction mixturewas irradiated with medium wavelength (302 nm) ultraviolet light and theamplified product was visualized by means of the associatedfluorescence. The fluorescence from the reaction mixture which containedthe nv(+)DNA was compared with that from a control reaction mixture,which was prepared in the same way as the nv(+)DNA reaction mixture froma control sample, that was the same as the sample with the nv(+)DNAexcept that it lacked the nv(+)DNA.

The results indicated at least 100-fold more fluorescent material in thereaction mixture prepared with the nv(+)DNA-containing sample. Thisquanitative difference was determined by comparison of the fluorescencefrom the reaction mixtures with fluorescent standards consisting ofdiluted samples of herring sperm DNA analyzed under identicalconditions.

Example 2

The product of the amplification reaction described in Example 1 wasalso analyzed by electrophoresis on an 8% polyacrylamide, 7M ureadenaturing gel according to the following procedure. A 30 ml gel (0.4 mmthick) was prepared by mixing 76 g/l acrylamide, 8 g/l bis-acrylamide,440 g/l urea, 500 μl/l TEMED in 1×TBE (1×TBE: 89 mM Tris base, 89 mMboric acid, 2 mM EDTA). To 30 ml of this solution was added 500 μl of afresh solution of 10% ammonium persulfate. After gel polymerization, 5μl samples of the nv(+)DNA amplification reaction mixture (whichincluded 25 attomoles of nv(+) DNA) were treated, prior to loading onthe gel, by being heated to 95° C. for 2 minutes in 20 μl blue juice(blue juice: 600 mg/ml urea, 1 mM EDTA, 5% glycerol, 0.05% bromophenolblue, 0.05% xylene cyanol). The gel was prerun for 30 minutes at 300volts and, after loading of samples, was run at 400 volts for 1 hour.The gel was then stained for 20 minutes in a solution of 0.5 μg/mlethidium bromide and nucleic acids were visualized by fluorescence,caused by exposure of the gel to ultraviolet light at 302 nm.

The results indicated that the product of the amplification reactionmigrated as a single band at a position consistent with theamplification of a 90-base RNA. Because the product fluoresced in thepresence of ethidium bromide and was synthesized using ribonucleosidetriphosphates, it must have been RNA. The original DNA material prior toamplification (which would have been present at less than 1 pg in thesolution as loaded on the gel) would not have been visible by thisprocess.

The procedure for determining that the amplified material included bothstrands of RNA was as follows. Two stained gels were prepared asdescribed above. Each was soaked for 20 minutes in 0.1 ×TBE containing0.5 μg/ml ethidium bromide and the nucleic acid electrophoreticallytransferred to a Hybond membrane filter (Amersham, Cat. No. RPN. 203N,Arlington Heights, Ill., USA) for 10 minutes at 50 volts in 0.1×TBE. Thefilter was washed for 30 minutes at 65° C. in 0.1×SSC (1×SSC: 150 mMsodium chloride, 15 mM sodium citrate), 0.5% SDS. The filter was thenprehybridized for 3 hours at 65° C. in 5×SSC, 40 mM NaPO₄, 5×Denhardt'sSolution (1×Denhardt's Solution: 200 μg/ml each of ficoll,polyvinylpyrrolidone, and bovine serum albumin (BSA)), 0.1 mg/ml shearedand denatured herring sperm DNA, and 10% dextran sulfate. ³²PO₄-kinasedoligonucleotide PM618 (a 66-base DNA probe with the sequence of SEQ IDNO: 17, which is the same as that of the 66 bases at the 5′-end ofnv(+)DNA) and ³²PO₄-kiased oligonucleotide PM624 (a 66-base DNA probewith the sequence of SEQ ID NO: 18, which is complementary to thesequence of the 66 bases at the 3′-end of nv(+)DNA), respectively, wereheated to 95° C. for 5 minutes and added for hybridization to theseparate filters that had been prepared, washed and prehybridized asdescribed, supra, (except that the filter used with PM618 had beenpreviously used with another probe buT this probe had been stripped byexposure of the filter to three sequential 1 minute treatments at 100°C. with 0.1×SSC, 0.1% SDS). The mixtures were hybridized overnight at65° C. Then, the filters were rinsed briefly with 2×SSC, 0.1% SDS, againwith the same solution at room temperature for 15 minutes and again for30 minutes. The filters were rinsed two more times for 30 minutes eachat 65° C. in 0.1×SSC, 0.1% SDS and were exposed to Kodak XAR-5 film at−80° C. using two DuPont Cronex Hi Plus intensifier screens.

The results indicated strong hybridization of both the PM618 and PM624to the products of the amplification reaction seen by ethidium staindescribed above. This indicates that both strands of nvRNA are made,fulfilling the final requirement of autocatalytic replication.

Additional support for the fact that DNA is amplified in the sample isderived from the following procedure, which eliminated RNA from thesample prior to amplification. One picomole each of nv(+)DNA andnv(+)RNA (the nanovariant RNA with the same sequence as nv(+)DNA) wereincubated for various time periods, in parallel but separately, in 1 mlof 1 N NaOH at 80° C. The treatments with alkali were followed byneutralization with an equal volume of 1 N HCl, buffering by addition ofTris·HCl, pH 7.5 to a final concentration of 460 mM, and dilution suchthat, if no template had been degraded in the alkali treatment, templatewould have been present at one attomole per 10 μl. Then, amplificationand detection procedures were carried out as described above. After 15minutes of alkali treatment, nv(+)RNA was no longer amplifiable, asindicated by the lack of fluorescent material found after theamplification procedure was carried out. However, nv(+)DNA wasamplifiable, after 15 or 60 minutes of alkali treatment, as indicated byat least a 100-times greater fluorescence intensity from the reactionmixture after the amplification procedure in comparison with suchintensity from control samples lacking any template and the sampleswhich contained only nv(+) RNA as template. After 180 minutes of thealkali treatment, the ability of nv(+)DNA to function as a template foramplification was also destroyed. Milder treatment of 15 minutes at 0.2N NaOH at 37° C. can also be used to discriminate amplification ofnv(+)RNA (or nv(−)RNA) template from that of nv(+)DNA (or nv(−)DNA)template.

An alternative treatment to destroy RNA present in samples, andtherefore diminish autocatalytically replicatable RNA as template foramplification by an RNA replicase, without degrading DNA, including suchDNA that might serve as a template for DDRP activity of such areplicase, is to treat with the nuclease RNAse A (10 μg/ml) for 20minutes at 37° C. This is followed by addition of 200 units of RNasin®RNase inhibitor (Promega Corporation, Madison, Wis., USA) to neutralizethe nuclease. For example, nv(+)DNA or nv(−)DNA samples treated in thismanner retain their ability to be amplified via the DDRP activity of Qβreplicase.

Example 3

This example is directed to illustrating additional oligonucleotidesingle-stranded and double-stranded templates for amplification by Qβreplicase via its DNA-dependent RNA polymerase activity.

The amplification procedures of Example 1 are followed, except that thetemplates described in Table 1 are used in the quantities described inTable 1, and are amplified under the time and temperature conditionsdescribed in Table 1. Except for herring sperm and stool DNAs;quantities are in attomoles. The templates listed, other than thosewhich are part of a plasmid, are single-stranded oligonucleotides. Thenucleotide sequences of the individual templates, other than theplasmids and the herring sperm and stool DNAs, are described in theSequence Listing. The “nv plasmid” is plasmid pNV-1-3-4 (see FIG. 8 andExample 6); in the linearized form, the plasmid was cut with arestriction endonuclease outside the segment of nanovariant DNA. The“description” is with reference to one strand, as indicated (or bothstrands, in the case of the nv plasmid) of nvDNA (see Example 1). Theamplified material is placed in a microtiter dish and is visualized asdescribed in Example 1. Amplification is indicated by a “+” sign, whilelack of amplification is indicated by a “−” sign.

TABLE 1 Template Template Assay Conditions Sequence Length Quantity TimeTemperature Amplification ID NO bases Strand Description attomoles min.° C. Results  1 90 + Full length 50 60 30 +  2 88 + 3′-deietion 50 3037 +  3 78 + Internal deletion 50 30 37 +  4 67 + 3′-deletion 50 60 30 + 5 87 + 5′-deletion 50 60 37 +  6 90 − Full length 50 60 30 +  7 129  −3′-extension 50 60 30 +  8 70 − 3′-deletion 50 60 30 +  9 119  +Insertion 50 60 30 + 10 138  + Insertion 50 60 30 + nv plasmid BothExtensions 1 30 30 + supercoil nv plasmid Both Extensions 1 30 30 +linear herring sperm DNA None No nv present   1 ng 30 30 − purifiedstool DNA None No nv present 0.01 ng 60 60 −

Example 4

This example illustrates a ligation/amplification procedure. One μl of200 mM NaCl containing 100 femtomoles of oligonucleotide PM754, 100femtomoles of target nucleic acid PM2123, and 50 femtomoles ofoligonucleotide PM2004 (see Sequence Listing for SEQ ID NOs 11, 21, 20for the sequences of PM754, PM2123, and PM2004, respectively) was mixedwith 2μl 10×ligase buffer (10×ligase buffer: 400 mM Tris·HCl, 100 mMMgCl₂, 10 mM DTT, 500 μg/ml acetylated BSA). The mixture was set at 70°C. and slow-cooled for 40 minutes to allow hybridization of theoligonucleotides to occur. 11 μl water, 2 μof a mix of 10 mM of eachrNTP, and 1 μl T4 DNA ligase (2 units) were added and the entire mixturewas set at 25° C. for 60 minutes. Two microliters of Qβ replicase (1.2mg/ml) was added and amplification proceeded for 30 minutes at 30° C.The reaction was terminated by transfer of the entire sample into 20μlof 2×stop solution (see Example 1). The products of the reaction werevisualized as described in Example 1. The products appeared as brightfluorescing material in microtiter wells. If the target molecule,PM2123, was left out of the reaction, fluorescence was very weak andsimilar to that observed in wells containing buffers but neither Qβreplicase nor probes.

The reaction products were analyzed following electrophoresis through an8% polyacrylamide 7M urea denaturing gel according to the followingprocedure. A 40 ml gel (1.5 mm thick) was prepared by mixing 76 g/lacrylamide, 4 g/l bis-acrylamide, 500 g/l urea in 1×TBE. To 50 ml ofthis solution were added 25 μl of TEMED and 250 μl of a fresh solutionof 10% ammonium persulfate. After gel polymerization, 5 μl samples ofamplification reaction mixtures were prepared by heating to 95° C. for 1minute in 25 μl blue juice (Example 2). The gel was prerun for 30minutes at 30 mA and, after loading samples, was run at 30 mA for 1.5hours. The gel was stained and visualized as described in Example 2.When the target, PM2123, was present in the reaction, two major bands,of approximately 118 and 110 bases in length, and at least 7 weakerbands, with lengths from about 80 to several hundred bases, wereobserved. These data indicate that the amplification of reportermolecules was dependent on the presence of target molecules in thesample.

Confirmation of target-specific amplification was demonstrated byhybridization of the electrophoretically separated material with probePM407 according to the following procedure, For the sequence of PM407,see SEQ ID NO: 19 in the Sequence Listing. Nucleic acid from the stainedgel was electrophoretically transferred to a Hybond membrane filter for20 minutes at 45 volts in 0.1×TBE. The resulting filter wasprehybridized for 1 hour at 65° C. as described in Example 2.³²PO₄-kinased oligonucleotide PM407 was added and the mixture washybridized for 4 hours at 60° C. The filter was rinsed for one minute atroom temperature in 2×SSC, 0.1% SDS and 5 times for 15 minutes each at60° C. in 2×SSC, 0.1% SDS. The resulting filter was exposed to KodakXAR-5 film at −80° C. using two DuPont Cronex Hi-Plus YE intensifierscreens. Hybridization was observed only with the 118-base band.

In different experiments with target nucleic acid present, products ofdifferent lengths, from several tens to several hundred bases, anddifferent distributions of amplification products among the variouslengths, have been observed. It has been found that, in a givenexperiment, some of the products include a segment with the sequencecomplementary to that of target segment, as judged by hybridization withPM407, and some do not. However, in every experiment when target,PM2123, was present, at least some of the product included a segmentwith the sequence complementary to that of target segment. Further, inexperiments in which PM2123 was not present, no reaction product thathybridized with PM407 was found.

Example 5

This example illustrates a target-dependent amplification processmediated by the DDRP activity of Qβ replicase following hybridization,but no ligation, of two probes which hybridize to adjacent segments oftarget nucleic acid and which both comprise a part of a segment of DNAwhich has the sequence of an RNA that is autocatalytically replicatableby the replicase. One microliter of 200 mM NaCl containing 100femtomoles PM754, 100 femtomoles PM2123, and 50 femtomoles PM2004 wasmixed with 2 μl 10×ligase buffer. The mixture was set at 70° C. andslow-cooled for 40 minutes to allow hybridization of theoligonucleotides to occur. Twelve microliters water, and 2 μl of a mixof 10 mM of each rNTP were added and the entire mixture was set at 25°C. for 60 minutes. Two microliters of Qβ replicase (1.2 mg/ml) was addedand amplification proceeded for 30 minutes at 30° C. The reaction wasterminated by transfer of the entire sample into 20 μl of 2×stopsolution (see Example 1). The products of the reaction were visualizedas described in Example 1.

The products appeared as bright fluorescing material in microtiterwells. If the target molecule, PM2123, was left out of the reaction,fluorescence was very weak and similar to that observed in wellscontaining buffers but neither the replicase nor probes. These dataindicate that the amplification of reporter molecules was dependent onthe presence of target molecules in the sample but did not requireligation of the probes hybridized adjacent one another on the target.The reaction products were analyzed following electrophoresis through apolyacrylamide-urea denaturing gel as described in Example 4. WhenPM2123 was present in the reaction, a single major band of approximately90 bases in length was observed. Confirmation of target-specificamplification was demonstrated by hybridization of theelectrophoretically separated material with probe PM407 as described inExample 4. Hybridization was observed only in the case of the 90-basereaction product generated in the presence of PM2123.

Example 6

This example illustrates the hybridization/separation/amplificationprocedure.

The target was a 107 nucleotide sequence of the E. coli lacZ gene whichcodes for a region at the amino terminus of the beta-galactosidaseprotein. The target region of the lacZ gene is contained in M13mp19phage DNA (Yanisch-Perron, C., et al. (1985), Gene 33:103-119). DNA fromthe related phage, φX174, was used as a negative control. Phage φX174DNA does not contain the lacZ gene.

The probe used in this example is isolated from the plasmid pNV1-3-4,which is illustrated in FIG. 8. pNV1-3-4 is a derivative of plasmidpUC18 (Yanisch-Perron, C., et al. (1985), Gene 33:103-119) and wasconstructed using standard techniques by replacing the small PstI-KpnIfragment of the polylinker of pUC18 with a segment providing the T7 RNApolymerase promoter and an nvDNA (double-stranded). The sequence of thispromoter/nvDNA-containing segment is shown in FIG. 8. The nvDNA segmentis indicated in the Figure as “nanovariant (+) strand,” because thesequence of the nv(+)DNA is shown. Plasmid pNV1-3-4 carries, within aPvuII/SmaI restriction fragment, both a segment with the sequencecomplementary to the 107 base target site, described above, and thenvDNA segment.

The probe was prepared by sequential digestion of plasmid pNV1-3-4DNAwith restriction endonucleases PvuII and Smal, respectively.Approximately 56 micrograms of plasmid DNA was digested with 140 unitsof PvuII (Promega, Madison, Wis., USA) at 37° C. in SmaI digestionbuffer (Promega) for 75 minutes in a final volume of 1 ml. The reactionwas cooled to 25° C., 200 units of SmaI was added, and the digestion wasallowed to continue for 5 hours at 25° C.

Approximately 13 μg (7 picomoles) of the digested DNA wasdephosphorylated and labeled at the 5′ terminus with ³²P-ATP (3,000Ci/mmole) using the reagents and conditions from the DNA 5′ End LabelingKit (Cat. No. 702757) from Boehringer Mannheim (Indianapolis, Ind.,USA). The three labeled fragments (2.37 kb, 214 bp, and 194 bp) wereseparated on a 10% polyacrylamide/7M urea gel (see Example 2). The 214bp fragment which contains the Qβ nvDNA segment, a T7 RNA polymerasepromoter segment, and the 107 bp complementary to the target in the lacZgene, was excised from the gel. The DNA was recovered from the gel by amodified “crush/elusion” method in which the gel fragment was placed ina LID/X test tube (LID/X Filter Syringe AQOR25, Genex, Gaithersburg,Md., USA) containing 0.4 ml of 100 mM NaCl, 0.1% SDS, 10 mM Tris·HCl, 1mM EDTA, pH 8. The tube was sealed with the filter-plunger and mixedovernight at 37° C. The filtrate was recovered and 0.4 ml of freshbuffer was added to the filter syringe, mixed for 2 hours at roomtemperature and filtered again. The filtrates were combined and theprobe concentration was determined by scintillation counting.

Approximately 2 picomols (0.3 ml) of the probe was mixed with 1 picomol(1 μl) of the target (or φX174, the negative control) and the mixturewas ethanol precipitated. The resulting DNA pellet was dissolved in 0.1ml of 2×SSC, 0.1% SDS. The probe and target (or negative control) weresubjected to hybridization conditions by heating to 100° C. for fiveminutes, followed by slow cooling to 50° C., and maintaining thetemperature at 50° C. for 90 minutes.

Following hybridization, the unhybridized probe was separated from phageDNA and hybrid molecules (i.e., probe-target hybrids) by gel filtrationon a Bio-Gel A-5 (BioRad, Richmond, Calif., USA) column (1 cm×28 cm) in100 mM NaCl, 10 mM Tris·HCl, 1 mM EDTA, pH 8.0. Eighty fractions (fivedrops each) were collected. The elution position of the phage DNA andthe unhybridized probe were determined by separate chromatographic runs.The elution position of the phage DNA was determined by a fluorometricDNA assay using Hoechst 33258 (bis-benzimide) dye (Boehringer-Mannhein,Indianapolis, Ind.) (0.15 μg/ml dye in 150 mM NaCl, 10 mM Hepes, pH 7.5;excitation at 354 nm; emission at 454 nm; with a Perkin-Elmer LS-3Fluorescence Spectrophotometer) from 40 μl aliquots of the columnfractions. The elution position of the unhybridized DNA probe wasdetermined by detection of either the ³²P-label on the probe or the Qβreplication products amplified from the nvDNA segment within the probe.

Referring now to FIGS. 9(a) and 9(b), the X-axes indicate fractionnumbers from the columns and the Y-axes indicate the amount ofradioactivity (cpm) present in each fraction. Particular fractions whichwere shown to amplify material in the presence of Qβ replicase areindicated by a plus (+) while those which were shown not to amplifymaterial in the presence of Qβ replicase are indicated by a minus (−).FIG. 9a illustrates the results of hybridization with the fragmentcontaining the target, and FIG. 9b shows the result of hybridizationwith the fragment which does not contain target.

Hybridization of probe to the lacZ gene in M13mp19 was indicated by thepresence of the probe in the M13mp19 DNA peak and was determined bydetection of the radioactive label and by the presence of DNAamplifiable by Qβ replicase as described in the next followingparagraph.

Following separation of unhybridized probe from phage DNA and hybridmolecules, respectively, samples with φX174 DNA and with M13mp19 DNA(including hybrid molecules) were combined with ribonucleosidetriphosphates and Qβ replicase for amplification in accordance with theprocedure described in Example 1, except that products fromamplification were measured with with a Perkin-Elmer fluorescencespectrophotometer measuring ethidium bromide fluorescence (0.5 μg/ml dyein water; excitation at 530 nm; emission at 600 nm).

Referring now to Table 2 below it will be seen that after hybridizationwith M13mp19 phage DNA, the hybrid DNA peak fraction contained 1230 cpmand produced 1485 fluorescence units of RNA after Qβ amplification. Thespecificity of the hybridization and detection steps were confirmed bythe use of a nonhomologous mock target DNA (φX174 phage DNA). The peakthat would have contained hybrid that was eluted from the Bio Gel A-5column after hybridization with φX174 DNA contained only a backgroundlevel of radioactively-labeled probe and no detectable RNA was producedby Qβ amplification.

TABLE 2 Fluorescence of Elution CPM from Qβ replicase Position ³²P-Probeproducts Controls M13mp19 Phage Peak 23 −1.5 Probe Peak 480 993.5 AssayBackground 20 −1.0 After Hybridization with M13mp19 phage DNA Phage Peak1230 1485 Probe Peak 1033 1033 After Hybridization with φX174 phage DNAPhage Peak 27 0.4 Probe Peak 476 1369

Example 7

This example illustrates the cDNA synthesis/amplification procedure. Inthis example, the probe is (+) strand nvRNA while the target is a21-base (−) strand DNA sequence which is complementary to the3′-terminal 21 bases of the (+) strand nvRNA. Seventy-five picomoles ofprobe was mixed with either 750 picomoles, 75 picomoles, 800 femtomoles,8 femtomoles, 80 attomoles, 800 tipomoles, 8 tipomoles, or 0 moles,respectively, of target in 5 μl of 1×SSC (150 mM NaCl, 15 mM sodiumcitrate) at 70° C. for 5 minutes to stimulate hybridization between thetarget and probe sequences. One microliter of this hybridization wasdiluted into a 20μl reaction mixture with final concentrations of 50 mMTris·HCl , pH 8.3, 7.5 mM NaCl, 0.75 mM sodium citrate, 19 mM KCl, 10 mMMgCl₂, 10 mM DTT, 1 mM each dNTP and 2.2 units AMV reversetranscriptase/μl and incubated for 1 hour at 42° C. to synthesize a cDNAcopy of the original RNA probe. The original RNA probe was destroyed bycombining 9 μl of this mixture with 100 μl of 1 N NaOH at 90° C. for 15minutes and then chilling the mixture on ice. The pH of the solution wasneutralized by addition of 100 μl of 1 N HCl. Four microliters of thisRNA-free cDNA solution were transferred to an amplification mixcontaining 100 mM Tris·HCl, pH 7.5, 100 mM NaCl, 15 mM MgCl₂1 mM each ofthe four ribonucleoside triphosphates (i.e., rNTPs) ATP, GTP, UTP, andCTP, and 100 μg/ml Qβ replicase. The mixture was incubated at 30° C. for60 minutes. The reaction products were visualized after denaturingpolyacrylamide gel electrophoresis as described in Example 2.

Samples containing greater than or equal to 20 attomoles of target inthe Qβ replicase reaction (which represents 8 femtomoles of target inthe hybridization step) were detected by this method. Hybridizationcontaining RNA probe with no target DNA produced no detectable productsignal using this method.

Example 8

This example illustrates a ligationlamplification 30 procedure usingpurified Salmonella genomic DNA as target. The oligonucleotides PM1059(with the sequence of SEQ ID NO: 24; compare with SEQ ID NO: 11 for thedecanucleotide added at the 5′-end of PM754 to make PM1059) and PM764(with sequence of SEQ ID NO: 12) were brought together by hybridizationto adjacent sequences of Salmonella DNA and were ligated in atarget-specific manner.

Oligonucleotide PM1059 was covalently attached at its 5′ terminus toparamagnetic particles (Advanced Magnetics, Cat. No. 4100B, Cambridge,Mass., USA). Thirty microliters (30 μg) of PM1059 particles wereconcentrated for one minute using a magnetic concentrator (DYNAL,Catalog No. MPCE, Oslo, Norway) and were resuspended in 48 μl ofhybridization solution (5×SSC, 1% BSA, 2% dextran sulfate, 0.1% TritonX-100) and prehybridized at 55° C. for 15 minutes. After 15 minutes, 1μl (containing 1 femtomole) of PM764 and 2 μl (containing 330 ng or 100am) of purified, denatured (by boiling for 5 minute) DNA from Salmonellatyphimurium (ATCC No. 14028) was added to the hybridization solution.The hybridization proceeded for 1 hour at 55° C. After hybridization,the particles were magnetically concentrated for one minute and werewashed twice with 2×SSC, 0.1% Triton X-100. Each wash involved adding200 μl of wash solution, vortexing briefly to resuspend the particles,magnetically concentrating the PM1059 particles for one minute, andremoving the wash solution. After removal of the second wash solution,the particles were resuspended in 50 μl of ligation/amplification buffer(ligation/amplification buffer: 40 mM Tris·HCl pH 7.8, 10 mM MgCl₂, 10mM dithiothreitol, 100 μl/ml bovine serum albumin, 500 nM ATP, and 1 mMof each of the four rNTPs) and 5 Weiss units of T4 DNA ligase and wereincubated at 30° C. for 1 hour. The ligated material was then amplifiedby the addition of 5 μl of Qβ replicase (1 mg/ml) to the ligationreaction and incubated at 30° C. for 1 hour. The reaction was terminatedby adding 55 μl of 2×stop solution to the amplification reactionmixture.

The products of the amplification were analyzed on an 8% denaturingpolyacrylamide gel as described in Example 2. The separated productswere electrophoretically transferred to a Hybond nylon filter(Amershain, Cat. No. RPN. 203N) for 20 minutes at 40 volts in 0.1×TBE.The filter was visualized under ultraviolet light at 302 nm to confirmtransfer of the stained products. RNA products on the filter werecross-linked to the filter by exposing the filter to 1200 μJ ofultraviolet light at 254 nm using a Stratalinker 1800 (Stratagene, Cat.No. 400071, La Jolla, Calif., USA). The filter was then prehybridizedfor one hour at 65° C. in 20 ml of hybridization solution B (5×SSC, 10%dextran sulfate, 100 μg/ml denatured herring sperm DNA, 40 mM NaPO₄, and5×Denhardt's Solution). The probe for this hybridization was PM407 (seeTable 1), with the sequence of SEQ ID NO: 19. Oligonucleotide PM407corresponds to the Salmonella sequence present in oligonucleotidePM1059. Hybridization with this probe indicates presence of amplifiedligated products because PM1059, alone, is not ampliflable. Probe PM407was kinase labelled with ³²PO₄ for 1 hour at 37° C. in a 10 μl volume.After heat killing the kinase at 90° C. for three minutes, the entirelabelling reaction mixture was added to the hybridization solution andfilter. The hybridization proceeded for four hours at 60° C. The filterwas rinsed briefly with wash solution (2×SSC, 0.1% SDS) at roomtemperature followed by 5 15-minute washes with wash solution at 60° C.The filter was then exposed to Kodak XAR-5 film at -80° C. for 16 hoursusing two DuPont Cronex Lightning Plus intensifier screens.

The results indicate that PM407 hybridized to an RNA product that wasapproximately 120 bp in size. In a parallel ligation/amplificationreaction in which the Salmonella target nucleic acid was not included,hybridization of the amplified products with the probe was not observed.This indicates that target specific ligation/amplification had occurred.

Example 9

This is an example of midivariant DNA amplification. The template usedin this example, pMDV XhoI, is a double-stranded plasmid, pSP64 (Melton,D., et al. (1984) Nucl. Acids Res. 12:7035-7056), containing a segmentwith the sequence of a recombinant midivariant RNA (Mills, D. R., et al.(1978) Proc. Natl. Acad. Sci. U.S., 75,5334-5338) (FIG. 10). Thesequence of the 274 bp HindIII-PstI fragment of pMDV XhoI is given bySEQ ID NO: 22. This fragment includes the mvDNA segment (“midivariant”DNA), which is from and including base pair 35 to and including basepair 266 of the sequence in SEQ ID NO: 22 and which has the sequence ofa midivariant RNA (capable of being autocatalytically replicated by Qβreplicase) modified by an insertion of ten base pairs, CCTCGAGGAG, whichincludes an XhoI site, which is present at positions 66-75 of themidivariant sequence and positions 100-109 in SEQ ID NO: 22. Restrictionendonuclease digestion with Pst I or Sma I, respectively, cleavesplasmid pMDV XhoI at the sites indicated at FIG. 10 Substrates werepreincubated at 80° C. in 1 N NaOH for 15 minutes and neutralized byaddition of an equivalent amount of HCl prior to their inclusion inreplicase reactions to remove the potential for contamination with RNAtemplates. As a control experiment, a sample of each base-treated DNAtemplate was also subjected to DNase treatment by addition of 5 units ofRQ1 RNase-free DNase (Promega Corporation) for 60 minutes at 37° C.

Midivariant DNA-containing DNA served as a template for DNA-dependentRNA polymerization by Qβ replicase by addition of 1 femtomole oftemplate in a 25 μl reaction vessel containing the following:

100 mM Tris·HCl, pH 7.5;

15 mM MgCl₂;

1 mM each of the ribonucleoside triphosphates ATP, GTP, UTP, CTP;

20 μg/ml Qβ replicase (Promega Corporation)

After addition of 5 microcurie (6.25 picomoles) α-³²P-CTP (DuPontCompany, NEN Research Products, Boston, Mass.), the mixture wasincubated for 60 minutes at 37° C. Amplification was monitored byspotting a portion of the reaction on a GFF filter (Whatman, Maidstone,England) precipitating the synthesized RNA by immersion of the filter inice code 10% trichloroacetic acid/1% sodium pyrophosphate. The filterswere washed four times with ice cold 5% trichloroacetic acid and thencounted by liquid scintillation.

The results (Table 3) indicate the dependence of amplification on thepresence of midivariant DNA sequences. They also indicate that moleculeswhich have the standard 3′ terminus exposed (Sma I-digested material) orthose with the standard 3′ terminus embedded within other DNA sequences(Pst I-digested material) both serve as amplifiable templates. Inaddition, undigested plasmids which are predominantly supercoiled alsomake effective substrates.

TABLE 3 DNA-DEPENDENT AMPLIFICATION OF MIDIVARIANT SEQUENCES Picomolesof α-³²P-CTP incorporated Base-Treated DNAse Template Base-TreatedTreated pMDV XhoI (SmaI- 460 7 digested pMDV XhoI (PstI- 420 1 digested)pMDV XhoI 720 14 (undigested)

Example 10

This example is directed to the use of chimeric DNA-RNA templates foramplification via the DDRP activity of Qβ replicase.

The amplification and detection procedures of Example 1 were followed,except that 10, 1 or 0.1 tipomoles of the chimeric template, PM1070 (SEQID NO: 25), which has the same sequence as the nanovariant positivestrand DNA (SEQ ID NO: 1) except that the 3 bases at the 5′-terminus andthe 6 bases at the 3′-terminus are ribonucleotides, were amplified for60 minutes at 30° C. Amplification was observed reproducibly with 1 ormore tipomoles of the template and in some experiments carried out with0.1 tipomoles of the template. No amplification was observed in theabsence of template.

A second chimeric template, PM1500 (SEQ ID NO: 26), which has the samesequence as the nanovariant positive strand DNA sequence, except thatbases 38, 39, 68 and 69 are ribonucleotides and the DNA5′-ATAAGCGCCATTGATGTTGTCGCC-3′ is joined to the 3′-terminus of thenv(+)DNA was also amplified.

The amplification procedure of Example 1 was followed except that 10tipomoles of template, PM1500, was amplified for 60 minutes at 30° C. in40 mM Tris·HCl, 10 mM DTT, 13 mM MgCl₂, 1 mM each rNTP, and 100 μg/ml Qβreplicase. The amplified material was placed in a microtiter dish andvisualized as described in Example 1. Ten tipomoles of PM1500 amplifiedwhile there was no visible amplification in the absence of template.

Example 11

This example illustrates the sensitivity of amplification of nvDNA andnv-chimeric templates. A “chimeric” template is one which has bothribonucleotides and 2′-deoxyribonucleotides in its sequence.

Varying amounts of nvDNA (PM444) and nv-chimera (PM1070) were amplifiedfor either 30 minutes or 60 minutes at 30° C. in 70 mM Tris·HCl, pH 7.6,10 mM MgCl₂, 5 mM DTT (dithiothreitol), 1 mM of each rNTP, and 100 μg/mlQβ replicase in a 10 μgl volume. Reactions were stopped by the additionof an equal volume of 2×stop solution. The reaction medium wasirradiated and visualized as described in Example 1. The least amount ofeach template amplified under each condition is shown in Table 4.

TABLE 4 Template Time Sensitivity PM444 30 min 300 tipomoles PM444 60min 30 tipomoles PM1070 30 min ≦10 tipomoles PM1070 60 min 1 tipomole

Example 12

This example illustrates the sensitivity of amplification of an mdvDNAtemplate.

Varying amounts of mdvDNA (gel purified Pst I/Sma I fragment of pMDVXhoI, FIG. 10) were base-treated, amplified, and detected as describedin Example 9, except that amplification was performed at 30° C. Theresults are shown in Table 5.

TABLE 5 Tipomoles Picomoles of of CTP Template Incorporated 100,000 9201000 710 10 165 0.1 44 0 11

Example 13

This example illustrates the sensitivity of amplification of DNA andchimeric templates in the presence of manganese chloride.

Varying amounts of nvDNA (PM444) and nv-chimera (PM1070) were amplifiedfor either 30 minutes or 60 minutes at 30° C. in 70 mM Tris·HCl, pH 7.6,10 mM MgCl₂, 1 mM MnCl₂, 5 mM DTT, 1mM each rNTP, and 100 μg/ml Qβreplicase in a 10 μl volume. Reactions were stopped and visualized asdescribed in Example 11 The reaction products were analyzed followingelectrophoresis through a polyacrylamide-urea denaturing gel asdescribed in Example 2. In the presence of MnCl₂, amplification in theabsence of template occurs after 30 minute or 60 minute reactions.However, the reactions produce a mixture of nucleic acid products ofvarious sizes, which we refer to as “de novo” synthesis. See Biebricheret al. (1986) Nature 321, 89-91. When bona fide template is present andamplified, a product which migrates as a 90-base product is visibleabove the background of de novo synthesis. Confirmation oftemplate-specific amplification was demonstrated by hybridization of theelectrophoretically separated material with probe PM624 as described inExample 2. Hybridization was observed only in the cases where templatewas present and amplified. Hybridization did not occur with productsgenerated by de novo synthesis.

The least amount of each template consistently amplified under eachcondition is shown in Table 6.

TABLE 6 Template Time Sensitivity PM444 30 min 10 tipomoles PM444 60 min10 tipomoles PM1070 30 min 1 tipomoles PM1070 60 min 1 tipomoles

Similar results were obtained with 0.5 mM MnCl₂ in the reaction mixture.With 2 mM MnCl₂ in the reaction mixture, the minimum detectable amountof target that was consistently observable remained the same as, but theamount of 90-base, target-segment-containing product from theamplification (over the same length of time) was less than, thatobserved when 1 mM MnCl₂ was used. With 0.25 mM MnCl₂, little or noeffect on sensitivity or rate of production of the 90-base,target-segment-containing amplification product was observed, incomparison with the sensitivity and rate of-production when no MnCl₂ wasused.

Example 14

This example illustrates the sensitivity of amplification of nvDNAtemplate in the presence of cobalt chloride.

Varying amounts of nvDNA (PM444) were amplified for 30 minutes at 30° C.in 70 mM Tris·HCl, pH 7.6, 10 mM MgCl₂, 1 mM CoCl₂, 1 mM of each rNTP,and 100 μg/ml Qβ replicase in a 10 μl volume. Reactions were stopped andvisualized as described in Example 11. The reaction products wereanalyzed by electrophoresis through a polyacrylamide-urea denaturing gelas described in Example 2. In the presence of CoCl₂, amplification inthe absence of template occurs within 30 minutes, giving rise to amixture of nucleic acid products of various sizes due to de novosynthesis. When bona fide template is present and amplified, one or moreprominent products which migrate at positions corresponding to about 90bases are seen above the background of de novo synthesis. Confirmationof template-specific amplification was demonstrated by hybridization ofthe electrophoretically separated material with probe PM624 as describedin Example 2. Hybridization was observed only in the cases where atleast 1 tipomole of template was present. Hybridization did not occurwith products generated by de novo synthesis.

While the invention is described in the present specification withconsiderable specificity, those of skill in the art will recognize manyvariations and modifications of what has been described that remainwithin the spirit of the invention. It is intended that suchmodifications and variations also be encompassed by the invention asdescribed and claimed herein.

26 90 base pairs nucleic acid single linear 1 GGGGAAATCC TGTTACCAGGATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGA GGACACACCC GGATCTAGCC 80GGGTCAACCC 90 88 base pairs nucleic acid single linear 2 GGGGAAATCCTGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGA GGACACACCCGGATCTAGCC 80 GGGTCAAC 88 78 base pairs nucleic acid single linear 3GGGGAAATCC TGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGAGGACACACCC GGATCTAG 78 67 base pairs nucleic acid single linear 4GGGGAAATCC TGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGAGGACACA 67 87 base pairs nucleic acid single linear 5 GAAATCCTGTTACCAGGATA ACGGGGTTTT CTCACCTCTC 40 TACTCGAAAG TTAGAGAGGA CACACCCGGATCTAGCCGGG 80 TCAACCC 87 90 base pairs nucleic acid single linear 6GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT 40 TCGAGTAGAG AGGTGAGAAAACCCCGTTAT CCTGGTAACA 80 GGATTTCCCC 90 129 base pairs nucleic acidsingle linear 7 GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT 40TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CCTGGTTACA 80 GGATTTCCCC TATAGTGTCACCTAAATTTC ACCTCTGCCT 120 AATCATCTC 129 70 base pairs nucleic acidsingle linear 8 GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT 40TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT 70 119 base pairs nucleic acid singlelinear 9 GGGGAAATCC TGTTAGGATC CAGGATAACG GGGTTTTCTC 40 ACCTCTCTATCTAGGGCGAC AACATCAATG GCGCTTATAA 80 AGTTAGAGAG GACACACCCG GATCTAGCCGGGTCAACCC 119 138 base pairs nucleic acid single linear 10 GGGGAAATCCTGTAACCAGG ATAACGGGGT TTTCTCAATA 40 AGCGCCATTG ATGTTGTCGC CTTTGTACGGCATACGGCCT 80 AACCACCTCT CTACTCGAAA GTTAGAGAGG ACACACCCGG 120 ATCTAGCCGGGTCAACCC 138 61 base pairs nucleic acid single linear 11 GGGGAAATCCTGTAACCAGG ATAACGGGGT TTTCTCAATA 40 AGCGCCATTG ATGTTGTCGC C 61 77 basepairs nucleic acid single linear 12 TTTGTACGGC ATACGGCCTA ACCACCTCTCTACTCGAAAG 40 TTAGAGAGGA CACACCCGGA TCTAGCCGGG TCAACCC 77 61 base pairsnucleic acid single linear 13 GGGGAAATCC TGTTACCAGG ATAACGGGGTTTTCTCAGGT 40 CAACTGAACG CCCTGAGCTT T 61 57 base pairs nucleic acidsingle linear 14 ATAAGCGCCA TTGATGTTGT CGCCCCTCTC TACTCGAAAG 40TTAGAGAGGA CACACCC 57 48 base pairs nucleic acid single linear 15TGGTTAGGCC GTATGCCGTA CAAAGGCGAC AACATCAATG 40 GCGCTTAT 48 48 base pairsnucleic acid single linear 16 GGCGACAACA TCAATGGCGC TTATAAAGCTCAGGGCGTTC 40 AGTTGACC 48 66 base pairs nucleic acid single linear 17GGGGAAATCC TGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGAGGACAC 66 66 base pairs nucleic acid single linear 18 GGGTTGACCCGGCTAGATCC GGGTGTGTCC TCTCTAACTT 40 TCGAGTAGAG AGGTGAGAAA ACCCCG 66 24base pairs nucleic acid single linear 19 ATAAGCGCCA TTGATGTTGT CGCC 2457 base pairs nucleic acid single linear 20 TTTGTACGGC ATACGGCCTAACCACCTCTC TACTCGAAAG 40 TTAGAGAGGA CACACCC 57 48 base pairs nucleicacid single linear 21 TGGTTAGGCC GTATGCCGTA CAAAGGCGAC AACATCAATG 40GCGCTTAT 48 274 base pairs nucleic acid double linear Sequence is thatof HindIII-EcoRI fragment, thought to be 274 bp in length, of plasmidpMDV XhoI. Both strands of the segment between bases 35-266, inclusive,as indicated in the sequence, are Q_-replicase amplifiable. The “N′s” atbases 7 and 51 are, independently, either G or no base. The “NN” atbases 260 and 261 are GG, C or no bases. It is not known whether the Kat base 262 is a T or a G. 22 AAGCTTNGGC TGCAGTCTAA TACGACTCACTATAGGGGAC 40 CCCCCCGGAA NGGGGGGACG AGGTGCGGGC ACCTGCTACG 80 GGAGTTCGACCGTGACGAGC CTCGAGGAGT CACGGGCTAG 120 CGCTTTCGCG CTCTCCCAGG TGACGCCTCGTGAAGAGGCG 160 CGACCTTCGT GCGTTTCGGT GACGCACGAG AACCGCCACG 200CTGCTTCGCA GCGTGGCCCC TTCGCGCAGC CCGCTGCGCG 240 AGGTGACCCC CCGAAGGGGNNKTCCCGGGA ATTC 274 232 base pairs nucleic acid double linear Sequenceis that of a midivariant DNA, thought to be 232 bases in length (basepairs 35 - 266 of the DNA fragment described in SEQ ID NO 1. The “N” atbase 17 is either G or no base. The “NN” at bases 226 and 227 are GG, Cor no bases. It is not known whether the K at base 228 is a T or a G. 23GGGGACCCCC CCGGAANGGG GGGACGAGGT GCGGGCACCT 40 GCTACGGGAG TTCGACCGTGACGAGCCTCG AGGAGTCACG 80 GGCTAGCGCT TTCGCGCTCT CCCAGGTGAC GCCTCGTGAA 120GAGGCGCGAC CTTCGTGCGT TTCGGTGACG CACGAGAACC 160 GCCACGCTGC TTCGCAGCGTGGCCCCTTCG CGCAGCCCGC 200 TGCGCGAGGT GACCCCCCGA AGGGGNNKTC CC 232 71base pairs nucleic acid single linear 24 CCTAGTCCAA GGGGAAATCCTGTTACCAGG ATAACGGGGT 40 TTTCTCAATA AGCGCCATTG ATGTTGTCGC C 71 90 basepairs nucleic acid single linear The three nucleotides at the 5′-end andthe six nucleotides at the 3′-end are ribonucleotides. 25 GGGGAAATCCTGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGA GGACACACCCGGATCTAGCC 80 GGGTCAACCC 90 90 base pairs nucleic acid single linear Thenucleotides at positions 38, 39, 68 and 69 are ribonucleotides. 26GGGGAAATCC TGTTACCAGG ATAACGGGGT TTTCTCACCT 40 CTCTACTCGA AAGTTAGAGAGGACACACCC GGATCTAGCC 80 GGGTCAACCC 90

What is claimed is:
 1. A nucleic acid molecule comprising the sequenceset forth in the SEQ ID NO:, the SEQ ID NO: selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO:12, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:
 22. 2. Anucleic acid according to claim 1, wherein the molecule comprises thesequence set forth in SEQ ID NO:
 11. 3. A nucleic acid according toclaim 1, wherein the molecule comprises the sequence set forth in SEQ IDNO:
 20. 4. A nucleic acid according to claim 1, wherein the moleculecomprises the sequence set forth in SEQ ID NO:
 24. 5. A nucleic acidaccording to claim 1, wherein the molecule comprises the sequence setforth in SEQ ID NO:
 12. 6. A nucleic acid according to claim 1, whereinthe molecule comprises the sequence set forth in SEQ ID NO:
 7. 7. Anucleic acid according to claim 1, wherein the molecule comprises thesequence set forth in SEQ ID NO:
 9. 8. A nucleic acid according to claim1, wherein the molecule comprises the sequence set forth in SEQ ID NO:10.
 9. A nucleic acid according to claim 1, wherein the moleculecomprises the sequence set forth in SEQ ID NO;
 22. 10. A nucleic acidmolecule consisting essentially of the sequence set forth in the SEQ IDNO:, the SEQ ID NO: selected from the group consisting of SEQ ID NO: 11,SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 10, and SEQ ID NO:
 22. 11. A nucleic acid molecule selectedfrom the group of nucleic acid molecules consisting of SEQ ID NO: 11,SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 10, and SEQ ID NO: 22.